WO2021199031A1 - Method of analyzing a single-cell western blot chip - Google Patents

Abstract

The present invention provides improved methods for implementing and analyzing single cell western blot applications comprising the step of measuring the DNA content per well. This improvement enables the user to distinguish between those wells containing a single cell, wells containing two cells and wells containing no cells whatsoever to provide accurate analysis of true single cell western blots.

Description

METHOD OF ANALYZING A SINGLE-CELL WESTERN BLOT CHIP

FIELD OF THE INVENTION

The present invention relates to improved methods for implementing and analyzing single cell western blot applications comprising the step of measuring the DNA content per well. This improvement enables the user to distinguish between those wells containing a single cell, wells containing two cells and wells containing no cells whatsoever to provide accurate analysis of true single cell western blots.

BACKGROUND OF THE INVENTION

For many purposes it is desirable or necessary to analyze the presence or quantity of one or more proteins in a single cell.

Various systems are commercially available that provide a platform for single cell protein analysis by western blot analysis. For example, various systems for single cell Western blot (scWestem) analysis are sold under the tradename ProteinS imple^® including a representative example sold under the trademark MILO^® which uses a multiwell design having 6400 individual wells per plate. Typically, the diameter of the wells is adapted to accommodate single cells that are sown on these multiwell plates at a dilution that statistically should provide a single cell per well. However, in reality at least some wells will contain doublets (i.e., two cells per well) and many will contain no cells at all. This means that using the MILO^® system as instructed will not enable a user to provide true single cell analysis of proteins.

ProteinS imple^® products are protected under one or more of the following U.S. Patents: 5,985,121, 6,853,454, 6,909,459, 7,064,826, 7,166,202, 7,217,937, 7,307,721, 7,379,577, 7,605,919, 7,846,676, 7,859,664, 7,914,656, 7,935,308, 7,935,479, 7,935,489, 8,021,611,

8,102,528, D516,733, D524,948, and other issued and pending patents in the U.S. and other countries.

There remains an unresolved problem with commercially available technologies of single cell western blot analysis to ensure that the results obtained represent true single cells. SUMMARY OF THE INVENTION

In order to truly understand the heterogeneity of the various cell populations, it is essential to investigate cells at the single-cell level. A major new technical finding of the present disclosure is the effectiveness of DNA staining in detecting wells with single- versus double-cell occupancies. Until the present invention, occurrence of double cells occupancy was measured by expression of housing-keeping proteins such as b-Actin. However, expression of b-Actin showed a high degree of variance among cells, a finding that is consistent to prior reports using bacterial and mammalian cells. Thus, probing with antibodies that target b-Actin or potentially other housing-keeping proteins in scWestem is unlikely to be useful in discriminating the occupancy of cells in a chip well.

The present invention provides methods that enable true single cell Western blot (scWestem) analysis of proteins by simultaneously providing quantification of DNA. It is known that during single cell Western blot, some of the wells are occupied with two cell (double occupancy) or not occupied at all. It was found according to the teachings of the present invention that it is possible to distinguish between single and double cells occupancy by measuring DNA content of the well. This method for the first time provides resolution of DNA from single cells vs. doublets, to ensure that the results obtained from the western blot analysis reflect true single cell protein analysis. This method also allows detecting wells that are not occupied with cells.

The present invention provides a much more efficient way to the existing single cell Western blot. As the current methods for detecting double-cell occupancy are very inefficient, thus, it is suggested to dilute the cells to concentration of 100,000 cells/ml before loading them on the chip in order to avoid double occupancy. Consequently, typically up to 1000 cells are loaded on the scWestem chip. By allowing good detection of well with double-cell occupancy, and subsequent elimination of these wells from analysis, we succeeded to increase the number of cells that may be loaded on the chip up to about 3000 by increasing the concentration of loaded cell. It is possible, therefore, to obtain at least 3 times more information from one chip than in the current methods. In addition, as there is no need to use antibodies against house-keeping proteins, additional types of targets may be measured. We have exemplified the methods of the present invention to demonstrate that about 12% of somatostatin-positive and 5% of glucagon-positive pancreatic cells simultaneously express insulin, establishing the existence of double-hormonal cells in islets isolated from normal human donors. This effect could be shown only after assuring that no double-cell occupied well are included in the analysis. The methods disclosed herein should have wide ranging applications in the study of complex primary samples. They have a significant advantage over present methods by enabling true single cell analysis. Current methods aim to prevent the occurrence of doublets and are limited to the adjustment of physical properties, such as the concentration of cells in the suspension used and/or well dimensions. In contrast, methods set forth by the invention allow for true single cell analysis even in the presence of doublets and under conditions not thought to be possible beforehand.

In one embodiment, a method of analyzing a single cell Western blot (scWestem) chip is provided, the method comprising: a. preparing a cell suspension; b. seeding cells of the cell suspension into the wells of the scWestem chip; c. lysing the seeded cells; d. electrophoretically separating cell content on a gel; e. staining the DNA content in each well of the scWestem chip; f. for each well of the scWestem chip, receiving a respective first signal comprising DNA data; g. for each well of the scWestem chip, comparing the DNA data of the respective first signal to at least one predetermined threshold; h. for each well of the scWestem chip, determining, responsive to an outcome of the respective comparison, occupancy information of the respective well; and i. responsive to the determined occupancy information for each well of the scWestem chip, outputting chip data of the scWestem chip.

In one embodiment, the DNA staining is effected using a fluorescent labelling. In one further embodiment, the fluorescent label of DNA is DAPI or TOTO-1 iodide.

In another further embodiment, the respective DNA data for each well of the scWestem chip comprises a predetermined function of the fluorescence values from the respective well associated with the DNA staining. In one yet further embodiment, the predetermined function comprises a multiplication of: an area under the curve (AUC) of the outcome of a convolution of a signal to noise ratio (SNR) signal and a predetermined peak shape, the SNR signal being of fluorescent counts in relation to a distance from the center of the respective well; and a width of the outcome of the convolution. In one embodiment, the at least one predetermined threshold comprises a predetermined statistical attribute of the DNA data of the wells of the scWestern chip. In another embodiment, the at least one predetermined threshold comprises a plurality of predetermined statistical attributes, each predetermined statistical attribute associated with a respective one of a plurality of sets of adjacent wells of the scWestern chip. In one further embodiment, the predetermined statistical attribute comprises a median value.

In one embodiment, the at least one predetermined threshold comprises the lowest value of the DNA data of the wells of the scWestern chip. In another embodiment, the at least one predetermined threshold comprises the lowest value of the DNA data of each of a plurality of sets of adjacent wells of the scWestern chip.

In one embodiment, for each of the wells of the scWestern chip, the method further comprises, responsive to an outcome of the comparison to the at least one predetermined threshold, determining whether the respective well is occupied by more than one cell. In another embodiment, for each of the wells of the scWestern chip, the method further comprises, responsive to an outcome of the comparison to the at least one predetermined threshold, determining whether the respective well is occupied by at least one cell.

In one embodiment, the at least one predetermined threshold comprises at least one first predetermined threshold and at least one second predetermined threshold, wherein, for each of the wells of the scWestern chip, the method further comprises: determining, responsive to an outcome of the comparison to the at least one first predetermined threshold, whether the respective well is occupied by more than one cell; and determining, responsive to an outcome of the comparison to the at least one second predetermined threshold, whether the respective well is occupied by at least one cell.

In one further embodiment, the comparison to the first predetermined threshold is performed only for cells of the scWestern chip determined to be occupied by at least one cell. In antoher further embodiment, the method further comprises, for each of the wells of the scWestern chip determined to be occupied by at least one cell, subtracting from the DNA data of the respective well a respective value associated with the DNA data of at least one well determined to not be occupied by at least one cell. In one yet further embodiment, the at least one well not occupied by at least one cell comprises the two of the wells closest to the respective well. In another embodiment, the cell suspension comprises up to 800,000 cells/ml. In one further embodiment, the cell suspension comprises from 10,000 to 500,000 cells/ml.

In one embodiment, the chip data comprises the occupancy information of the wells of the scWestem chip. In another embodiment, the method further comprises immobilizing the proteins, peptides and/or polypeptides in the gel.

In one further embodiment, the method further comprising staining the immobilized proteins and/or peptides. In another further embodiment, the method further comprises receiving a second signal obtained from the staining.

In another further embodiment, the method further comprises for each of the wells of the scWestem chip responsive to the received second signal, measuring at least one peptide or protein in the respective well. In one yet further embodiment, measuring at least one peptide or protein comprises qualitative and quantitative measurement.

In one further embodiment, the output chip data comprises the second signal or the results of the measurement of the at least one peptide or protein. In another further embodiment, the method further comprises, responsive to the occupancy information of each of the wells of the scWestem chip, generating a dataset comprising the results of the measurement for a subset of the wells.

In one yet further embodiment, the subset of the wells excludes (i) wells that are not occupied by at least one cell; (ii) wells that are occupied by more than one cell; or (iii) both (i) and (ii). In another yet further embodiment, the chip data comprises the generated dataset.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates a high-level block diagram of a system for analyzing an scWestem chip, in accordance with some embodiments. Fig. 2 illustrates a high-level flow chart of a method of analyzing an scWestem chip, in accordance with some embodiments.

Fig. 3. shows the ability of DNA measurement in the well to differentiate since cell and doubles. Fig. 3A shows a representative raw image of a scanned Milo scWestem blot chip loaded with islet cells stained with antibodies against B-Actin is shown. Fig. 3B shows a fluorescence signal-to-noise ratio (SNR) as a factor of migration distance was measured in each well by the manufacture’s software and was shown as a curved line. Fig. 3C shows the histogram ranges of b-Actin expression levels. Fig. 3D shows representative photomicrographs of 2 wells of live cells under a microscope with visible-light illumination, lyzed, ran on scWestem, stained with a DNA dye, and imaged again with the DNA staining. Fig. 3E shows the AUCs of the DNA levels multiplied with the fluorescence SNR values in each wells (rectangles - high DNA content; circles - low DNA content). Fig. 3F shows the means of DNA-AUC measurements of the two groups, varied approximately by 2 fold, were significantly different as compared by the Student’s T-Test (****=p<0.0001).

Fig. 4. Shows quantification of hormone expression levels by beta, alpha and delta cells. Fig. 4A shows the experimental workflow. Figs. 4B, C and D shows shapes of SNR curves along the migration distance for INS, GCG and SST, respectively Fig. 4E-G show a histogram of hormone expression levels ranges (AUCs) for INS, GCG and SST, respectively

Fig. 5. shows the levels of hormone expression in single- and double-hormonal cells. The levels of expression for individual double-hormonal cells were normalized to the mean expression of single-hormonal cells. The mean expression was compared between single (GCG⁺/SST⁺) and double (GCG⁺INS⁺/SST⁺INS⁺) hormonal cells by 2-Way ANOVA (n=4 donors, * indicates p<0.05, **** indicates p<0.0001).

Fig. 6 shows the data collected from DNA content of wells on the scWestem chip and separation between wells comprising single or double occupancy based on the determined threshold.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention for the first time discloses methods for the performance of single cell western blot protein analysis while simultaneously enabling measuring DNA signal in an individual sample. This improvement allows for the first time true single cell analysis of proteins through verification of DNA content.

As exemplified herein below, true single cell samples were identified by determining single- or a double-cell (also referred to as doublets) well occupancy through measuring DNA data . This technique, and other aspects set forth by the invention, enabled more precise analysis of small peptide hormones than was previously feasible. The exemplary results of pancreas cell analysis permitted by aspects of the invention unequivocally demonstrate high variance of hormone levels in individual alpha, beta and delta cells. Importantly, about 12% of somatostatin-positive and 5% of glucagon-positive cells simultaneously express insulin, establishing the existence of double-hormonal cells in islets isolated from normal human donors. These methods have wide-application to the study of complex primary samples.

The present invention provides a method of determining the occupancy of wells of a single cell Western blot (scWestern) chip. The method comprises the steps of: a. preparing a cell suspension; b. seeding cells of the cell suspension into the wells of a scWestern chip; c. lysing the seeded cells; d. electrophoretically separating cell content on a gel; e. staining the DNA content in each well of a scWestern chip; f. measuring the signal obtained from the DNA staining for each well; g. calculating a threshold value; and h. determining the occupancy of each well based on a the signal obtained from the DNA staining in comparison to the threshold value.

According to one aspect, the present invention provides a method of analyzing a single cell Western blot (scWestern) chip, the method comprising: a. preparing a cell suspension; b. seeding cells of the cell suspension into the wells of the scWestern chip; c. lysing the seeded cells; d. electrophoretically separating cell content on a gel; e. staining the DNA content in each well of the scWestern chip; f. for each well of the scWestern chip, receiving a respective first signal comprising DNA data; g. for each well of the scWestern chip, comparing the DNA data of the respective first signal to at least one predetermined threshold; h. for each well of the scWestern chip, determining, responsive to an outcome of the respective comparison, occupancy information of the respective well; and i. responsive to the determined occupancy information for each well of the scWestern chip, outputting chip data of the scWestern chip.

As used herein, the term “occupancy” refers to existence of cells in a scWestern plate well following the seeding of a cell suspension into the wells of a scWestern plate. As used herein, the terms “double occupancy”, “double cells occupancy” and “doublet” are used herein interchangeably and refer to presence of 2 cells in the well of scWestem chip.

As used herein, the term “cell suspension” refers to a type of cell culture in which single cells or small aggregates of cells are allowed to function and multiply in an agitated growth medium, thus forming a suspension. According to some embodiments, the concentration of cells in the cell suspension is up to 1,000,000 cells/ml. According to other embodiments, the concentration of cells in the cell suspension is up to 800,000 cells/ml or up to 600,000 cells/ml. According to one embodiment, the concentration of cells in the cell suspension is up to 500,000 cells/ml. According to another embodiment, the concentration of cells in the cell suspension is up to 400,000 cells/ml. According to yet another embodiment, the concentration of cells in the cell suspension is up to 350,000 cells/ml. According to some embodiments, the concentration of cells in the cell suspension is up to 300,000 cells/ml. According to some embodiments, the concentration of cells in the cell suspension is up to 250,000 of up to 200,000 cells/ml. According to some embodiments, the concentration of cells in the cell suspension is from 10,000 to 1,000,000, from 20,000 to 800,000, from 30,000 to 700,000, from 40,000 to 600,000, from 50,000 to 500,000, from 60,000 to 450,000, from 70,000 to 400,000, from 80,000 to 350,000, from 90,000 to 300,000, from 100,000 to 250,000 from 120,000 to 200,000 cells/ml. According to some embodiments, the concentration of cells in the cell suspension is from 10,000 to 400,000 from 20,000 to 350,000, from 30,000 to 300,000, from 50,000 to 250,000, from 100,000 to 400,000, or from 100,000 to 300,000 cells/ml.

As used herein, the term “cell seeding” or the interchangeable term “cell sedimentation” refers to the passive gravity-driven settling of cells in the wells of a scWestem plate. Alternatively, the term may refer to any other force-driven sedimentation.

As used herein, the terms “scWestem plate” and “scWestem chip” are used herein interchangeably and refer to a slide micropattemed with an chip of wells, e.g. microscopic slide coated with a thin photoactive polyacrylamide gel (PAG) micropattemed with an chip of wells (i.e. microwells). scWestem plates may be prepared by the surface silanization of a standard glass microscope slide and the epoxy-based negative photoresist (SU-8) mold casting of microwells in a thin PAG layered on the glass microscope slide, to create either a PAGE gel of uniform pore size or a PAGE gel of gradient pore size. Any know scWestem plate may be used in the methods of the present invention, e.g. plate adapted for Milo™ single-cell western (scWestem) platform. Examples of such chips are scWest chip manufactured by ProteinS imple. According to some embodiments, the loaded chip comprises from 500 to 4000 cells. According to some embodiments, the loaded chip comprises from 600 to 3500, from 700 to 3000, from 800 to 2500 cells. According to some embodiment, the loaded chip comprises from 900 to 2400, from 1000 to 2000, from 1200 to 1800, or from 1000 to 2500 cells.

The term “chip data” refers to any data that may be obtained from the Western blot chip, including occupancy data per each well, protein data obtained from cells, and any processed data obtained from the chip, e.g. protein data obtained from a subset of well, said subset comprising only single-occupied wells.

As used herein, the term “PAGE” or “polyacrylamide gel electrophoresis” refers to a technique widely used in biology and biotechnology to separate proteins according to their electrophoretic mobility, and that is included in a step of the scWestem protocol.

As used herein, the term “immunoprobing” refers to incubating the gel with solutions of primary antibodies and then with fluorescently labeled secondary antibodies.

The term “fluorescence” is a well-known term referring to an optical phenomenon in which a molecule absorbs a high-energy photon and re-emits it as a lower-energy (longer- wavelength) photon, with the energy difference between the absorbed and emitted photons ending up as molecular vibrations or heat. The terms “fluorescence value”, “fluorescence count” and “fluorescence signal” may be used interchangeably and refer to the values obtained from measurement the fluorescence intensity. As used herein, the term “sum of fluorescence” refers to the area under the curve (AUC), calculated from a curve obtained by plotting the signal-to- noise ratio of a fluorescence signal along its migration distance.

The examination of cell-to-cell heterogeneity has contributed greatly to the understanding of cellular processes such as stem cell differentiation and development, immune response, pharmaceutical efficacy, and cancer. When studying the activity of complex cell populations, it is advantageous to use analytical tools offering true single-cell resolution that provide quantitative and highly specific detection of target proteins. Single-cell western blot (scWestem) analysis has emerged as a useful assay for rapid, sensitive, and selective quantitative analysis of protein expression. scWestem is performed on a microdevice that comprises an chip of wells molded in a thin layer of a polyacrylamide gel (PAG). scWestem involves five main steps: (i) gravity settling of cells into wells; (ii) chemical lysis of cells in each well; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins.

According to some embodiments, seeded cells are lysed. Any known methods for lysing cells can be implemented including chemical, enzymatically and physical, e.g. by sonication or homogenization. According to some embodiments, the cells are lysed according to the protocol of a device for single cell Western blotting.

According to some embodiments, the cell content is separated by electrophoresis on a gel or on the chip. Any known methods and techniques for performing electrophoresis may be used for that purpose. In certain embodiments, the gel is a polymeric gel. The polymeric gel may be a gel suitable for gel electrophoresis. The polymeric gel may include, but is not limited to, a polyacrylamide gel (e.g., methacrylamide gel), an agarose gel, and the like. The resolution of the separation medium may depend on various factors, such as, but not limited to, pore size, total polymer content (e.g., total acrylamide content), concentration of cross -linker, applied electric field, assay time, and the like. For instance, the resolution of the separation medium may depend on the pore size of the separation medium. In some cases, the pore size depends on the total polymer content of the separation medium and/or the concentration of cross-linker in the separation medium. In certain instances, the separation medium is configured to resolve analytes with molecular mass differences of 50,000 Da or less, or 25,000 Da or less, or 10,000 Da or less, such as 7,000 Da or less, including 5,000 Da or less, or 2,000 Da or less, or 1,000 Da or less, for example 500 Da or less, or 100 Da or less. In some cases, the separation medium may include a polyacrylamide gel that has a total acrylamide content, T (T=total concentration of acrylamide and bisacrylamide monomer), ranging from 1% to 20%, such as from 3% to 15%, including from 5% to 10%. In some instances, the separation medium has a total acrylamide content of 7.5%. In certain cases, the separation medium has a total acrylamide content of 6%.

In some embodiments of the method, the method further includes determining whether an analyte of interest is present in the sample. In certain cases, the determining includes contacting the analyte of interest with a label that specifically binds to the analyte to produce a labeled analyte.

In some embodiments of the method, the method further includes detecting the labeled analyte. In some embodiments, the analyte is the cell’s protein, peptide or polypeptide. Embodiments of the methods are directed to determining whether an analyte is present in a sample, e.g., determining the presence or absence of one or more analytes in a sample. In certain embodiments of the methods, the presence of one or more analytes in the sample may be determined qualitatively or quantitatively. Qualitative determination includes determinations in which a simple yes/no result with respect to the presence of an analyte in the sample is provided to a user. Quantitative determination includes both semi-quantitative determinations in which a rough scale result, e.g., low, medium, high, is provided to a user regarding the amount of analyte in the sample and fine scale results in which an exact measurement of the concentration of the analyte is provided to the user. According to some embodiments, the method of the present invention allows measuring the amount of the analyte.

As described above, the samples that may be assayed in the methods may include one or more analytes of interest. Examples of detectable analytes include, but are not limited to: nucleic acids, e.g., double or single-stranded DNA, double or single-stranded RNA, DNA-RNA hybrids, DNA aptamers, RNA aptamers, etc.; proteins and peptides, with or without modifications, e.g., antibodies, diabodies, Fab fragments, DNA or RNA binding proteins, phosphorylated proteins (phosphoproteomics), peptide aptamers, epitopes, and the like; small molecules such as inhibitors, activators, ligands, etc.; oligo or polysaccharides; mixtures thereof; and the like. In some embodiments, the analyte of interest can be identified so that the presence of the analyte of interest can then be detected. Analytes may be identified by any of the methods described herein. For example, the analyte may include a detectable label. Detectable labels include, but are not limited to, fluorescent labels, colorimetric labels, chemiluminescent labels, enzyme-linked reagents, multicolor reagents, avidin-streptavidin associated detection reagents, non-visible detectable labels (e.g., radiolabels, gold particles, magnetic labels, electrical readouts, density signals, etc.), and the like.

According to some embodiments, the method of the present invention comprises staining DNA content in each well of the scWestern chip. Obviously, the staining occurs only in well in which cells were present and lysed. Any known method for staining DNA may be used and a subsequently the corresponding methods for detecting the presence of DNA are utilized. According to some embodiments, DNA staining is effected using fluorescent labelling configured to label DNA. In certain embodiments, the detectable label is a fluorescent label. Fluorescent labels are labeling moieties that are detectable by a fluorescence detector. For example, binding of a fluorescent label to an analyte of interest such as DNA may allow the analyte of interest to be detected by a fluorescence detector. Examples of fluorescent labels include, but are not limited to, fluorescent molecules that emit fluoresce upon contact with a reagent, fluorescent molecules that emit fluoresce when irradiated with electromagnetic radiation (e.g., UV, visible light, x-rays, etc.), and the like. Suitable fluorescent molecules (fluorophores) include, but are not limited to, TOTO®-l iodide (Quinolinium, 1-1'-[1,3- propanediylbis [(dimethyliminio)-3 , 1 -propanediyl] ]bis[4- [(3-methyl-2(3H)- benzothiazolylidene)methyl]]-, tetraiodide 143413-84), fluorescein, fluorescein isothiocyanate, succinimidyl esters of carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer of fluorescein dichlorotriazine, caged carboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine, Texas Red, propidium iodide, JC- 1 (5,5',6,6'-tetrachloro-l ,1 ',3,3'- tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethyl rhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester), tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, cyan-shifted green fluorescent protein, red-shifted green fluorescent protein, yellow-shifted green fluorescent protein, 4- acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives, such as acridine, acridine isothiocyanate; 5-(2'- aminoethyl)aminonaphthalene- 1 -sulfonic acid (EDANS); 4-amino-N-[3- vinylsulfonyl)phenyl]naphth- alimide-3,5 disulfonate; N-(4-anilino- 1 -naphthyl)maleimide; anthranilamide; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5- indacene-3-propioni-c acid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives: coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4- trifluoromethylcoumarin (Coumarin 151 ); cyanine dyes; cyanosine; 4',6-diaminidino-2- phenylindole (DAPI); 5', 5"- dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2- ,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'- disulfonic acid; 5-(dimethylamino]naphthalene-l -sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate, erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives: 5- carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)amino- -fluorescein (DTAF), 2',7'dimethoxy-4'5'-dichloro-6- carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelli- feroneortho cresolphthalein; nitro tyro sine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 - pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 , sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6- carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-(4'- dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CAL Fluor orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine, coumarins and related dyes, xanthene dyes such as rhodols, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol, and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, fluorescent europium and terbium complexes; combinations thereof, and the like. Suitable fluorescent proteins and chromogenic proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a GFP derived from Aequoria victoria or a derivative thereof, e.g., a "humanized" derivative such as Enhanced GFP; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi; "humanized" recombinant GFP (hrGFP); any of a variety of fluorescent and colored proteins from Anthozoan species; combinations thereof; and the like. In certain embodiments, the method includes introducing a fluid sample into a microfluidic device. Introducing the fluid sample into the microfluidic device may include directing the sample through a separation medium to produce a separated sample. In some cases, the separated sample is produced by gel electrophoresis as the sample traverses the separation medium, as described above. The separated sample may include distinct detectable bands of analytes, where each band includes one or more analytes that have substantially similar properties, such as molecular weight, size, charge (e.g., charge to mass ratio), isoelectric point, etc. depending on the type of gel electrophoresis performed.

According to some embodiments, the DNA is stained by a fluorescent labeling. Any one of the abovementioned labels may be used. According to one embodiment, the fluorescent labeling comprises DAPI. According to another embodiments, the DNA is stained by TOTO-1 iodide. According to some embodiments, the fluorescent label is selected from DAPI, TOTO-1 iodide, Hoechst, 7- ADD, Crystal violet, and ethidium bromide. According to some embodiments, the fluorescence is detected and measured. According to some embodiments, the method comprises measuring the fluorescence signal obtained from DNA staining. According to some embodiments, the fluorescence measuring is effected after step (e).

In some embodiments of the method, the label includes a labeled antibody.

In some embodiments, the method includes the digital storage of images and pattern processing in a computer system for automated cell processing and analysis.

Fig. 1 illustrates a high-level block diagram of a system 10 for analyzing a scWestern chip, in accordance with some embodiments. System 10 comprises: a single cell Western blot platform 20, such as the Milo^® platform, including a microarray scanner; a processing system 30; and a memory 40. In one embodiment, processing system 30 and memory 40 are embedded as part of single cell Western blot platform 20. In one embodiment, memory 40 comprises instructions, which when run by one or more processors of processing system 30, cause processing system 30 to perform one or more methods, as described below in relation to stages 1010 - 1050.

Fig. 2 illustrates a high-level flow chart of a method of analyzing an scWestern chip, in accordance with some embodiments. In stage 1000, a cell suspension is prepared and the cells of the cell suspension are seeded into the wells of the scWestern chip. The seeded cells are then lysed and the cell content is electrophoretically separated on a gel or on the chip. The DNA content in each well of the chip is then stained with a predetermined DNA dye.

In stage 1010, for each of a plurality of wells of the chip of stage 1000, a respective first signal is received, the respective first signal comprising DNA data regarding the contents of the respective wells. The first signal is received from an output of an imager. The term "DNA data", as used herein, means data associated with DNA. In one embodiment, the DNA data of each respective first signal comprises a respective value. In another embodiment, the DNA data comprises a predetermined function of the fluorescence within a predetermined wavelength range associated with the predetermined DNA dye. Particularly, the predetermined wavelength range is a wavelength range that the fluorescence of the particular DNA dye is the strongest, as known to those skilled in the art.

In one embodiment, an SNR function is determined, the SNR function being a convolution of: a signal to noise ratio (SNR) signal of the fluorescent counts in relation to a distance from the center of the respective well; and a predetermined canonical peak shape. In one further embodiment, the canonical peak shape is an inverse parabola. In one embodiment, an area under the curve (AUC) of the SNR function is then determined. In one further embodiment, the predetermined function comprises a multiplication of the AUC of the SNR function and the width of the SNR function. In one embodiment, the AUC and the width of the SNR function are calculated between two local minimums of the fluorescence signal. Therefore, according to some embodiments, the DNA data comprises the AUC of the SNR function. In one further embodiment, the SNR function is determined by the Milo^® platform, as is the AUC and width thereof.

In stage 1020, for each of the plurality of wells of the chip of stage 1000, the DNA data of the respective first signal of stage 1010 is compared to at least one predetermined threshold. In one embodiment, the at least one predetermined threshold comprises at least one first threshold and at least one second threshold.

In one embodiment, the at least one first threshold comprises a predetermined statistical attribute of the DNA data. In one further embodiment, the statistical attribute comprises a mean of the values of the DNA data from one or more sets of wells of the chip. In one further embodiment, where a plurality of sets of wells of the chip are defined, each set of wells is defined as a predetermined number of adjacent wells. In one embodiment, there is a predetermined amount of overlap between adjacent sets of wells. Using a plurality of sets of wells reduces the amount of variability that is naturally present across the chip. In another embodiment, the DNA data values of each well of the chip is determined and each of the plurality of sets of wells is defined such that the variance of the DNA data values in each set is less than a predetermined maximum value. In such an embodiment, the different sets of wells can differ from each other in size and/or shape.

The difference between the value of the DNA data from each wells of the chip is the determined mean of the respective set of wells is then determined. In such an embodiment, the first predetermined threshold is determined as a maximum difference from the determined mean. In one further embodiment, the maximum difference is about 1.4 - 1.8 times the median value, preferably 1.5 times the median value. Although the above embodiment has described in relation to an embodiment where the statistical attribute comprises a mean, this is not meant to be limiting in any way. In another embodiment, the statistical attribute comprises the median, or other statistical function of the values of the DNA data from the one or more sets of wells of the chip. In one embodiment, the second predetermined threshold is zero. In another embodiment, for each of the one or more sets of wells, the lowest DNA value of the respective set is determined. In such an embodiment, the second predetermined threshold is the lowest DNA value, i.e. the value of the background.

In stage 1030, responsive to outcomes of the comparisons of stage 1020, occupancy information of each well is determined. In one embodiment, for each of the plurality of wells of the chip of stage 1000, responsive to an outcome of the comparison to the at least one first threshold of stage 1020, it is determined whether the respective well of the chip is occupied by more than one cell. Particularly, if the respective value of the DNA data is greater than the first predetermined threshold it is due to there being more than one cell in the respective well.

In another embodiment, for each of the plurality of wells of the chip of stage 1000, responsive to an outcome of the comparison to the at least one second predetermined threshold of stage 1020, it is determined whether the respective well of the chip is occupied by at least one cell. Particularly, if the respective value of the DNA data is greater than the second predetermined threshold, it is due to there being at least one cell in the respective well. Thus, the term "occupancy information", as used herein, means whether the respective well contains no cells, one cell or more than one cell.

In one embodiment, the comparison of the DNA data to the first predetermined threshold is performed only for cells that have been determined to be occupied by at least one cell.

In another embodiment, prior to the comparison of the DNA data to the first predetermined threshold, for identifying wells being occupied by more than one cell, the background noise is removed. In one embodiment, the background noise is determined for each well by determining the DNA data values of at least one well determined to not be occupied by at least one cell, i.e. wells without cells. In one further embodiment, the DNA data values of the two closest wells not occupied by any cells are determined. A predetermined function of the DNA data values is determined, thereby defining the background noise for the respective well. In one further embodiment, the predetermined function is an average. The background noise value is then subtracted from the DNA data of the respective well, and the subtracted value is compared to the respective first predetermined threshold. In such an embodiment, the first threshold is defined after subtracting the background values for each well. In optional stage 1040, the proteins, peptides and/or polypeptides in the gel of stage 1000 are immobilized. In such an embodiment, for each of the wells of the scWestem chip of stage 1000 a second signal is received. The second signal is received from the output of an imaging system. In one embodiment, the second signal differs from the first signal in that the fist signal is associated with imaging of a first wavelength range and the second signal is associated with imaging of a second wavelength range. Responsive to the received second signal, the presence of at least one target in the respective well is quantified, the target being a peptide, polypeptide and/or protein, as known to those skilled in the art of single cell Western blots.

In stage 1050, responsive to the determined occupancy information of stage 1030 for each well of the scWestem chip, chip data of the chip is output. In one embodiment, the chip data comprises the determined occupancy information, i.e. how many cells are in each well. In one embodiment, where the quantification of optional stage 1040 is performed, a dataset is generated. The dataset comprises the results of the quantification for a subset of the wells and the output chip data comprises the generated dataset. In one embodiment, the subset of the wells includes only wells which contain single cells, i.e. the subset of the wells for the dataset excludes wells that are not occupied by at least one cell and excludes wells that are occupied by more than one cell. In another embodiment, the subset of wells for the dataset includes only wells that contain at least one cell. In another embodiment, the subset of wells for the dataset excludes wells containing more than one cell.

According to any one of the above embodiments, the method further comprises immobilizing the cell content in the gel. According to some embodiments, the method further comprises immobilizing a target molecules in the gel. According to some embodiments, the target is selected from proteins, peptides and polypeptides in the gel. Thus, according to some embodiments, the method comprises immobilizing proteins, peptides and polypeptides in the gel. According to one embodiment, the method comprises immobilizing the cell proteome in the gel. According to another embodiment, the method comprises immobilizing the peptide content on the gel. According to yet another embodiments, the method comprises immobilizing the proteins and peptides in the gel. Any known method may be used. For example, the method comprises the procedure suggested by the manufacturer of the scWestem equipment, such as protocols of Milo™.

According to any one of the above embodiments, the method further comprises staining the proteins and/or the peptides immobilized in the gel. Any known method may be used for that purpose. Non-limiting examples are immuno-staining using antibodies (such as use of primary and secondary antibodies) and ELISA. According to some embodiments, the method comprises use of antibodies labelled with a fluorescent label.

According to some embodiments, the fluorescence from staining the target is detected and measured. According to some embodiments, the method comprises measuring the fluorescence signal obtained from protein, peptide and/or polypeptides staining. According to some embodiments, the second signal comprises the measurement of the fluorescence signal obtained from protein, peptide and/or polypeptides staining. According to some embodiments, the fluorescence signal obtained from protein, peptide and/or polypeptides staining is further processed.

According to any one of the above embodiments, the method further comprises measuring at least one target. According to some embodiments, the target comprises peptides and proteins. According to some embodiments, the measurement comprises qualitative or quantitative measurement. The methods for qualitative or quantitative measurement of proteins and peptides using staining, such as immuno-staining are well-known in the art. Any such method may be used. Thus, according to some embodiments, the method comprises detecting the presence of one or more proteins or peptides. According to other embodiments, the method comprises measurement the amount of at least one peptide or protein. According to some embodiments, measuring at least one target comprises comprise fluorescent measurement obtained from the immuno-staining. According to any one of the above embodiments, several targets may be measured. As described above, one of the advantages of the method of the present invention is that there is no need to measure housekeeping proteins. Considering that the source of primary antibodies is limited, the method allows measuring more targets. According to some embodiments, the second signal(s) is the signal obtained from proteins/peptides staining. According to some embodiments, the measured signal obtained from staining the peptides and/or the proteins, e.g. signal obtained from fluorescently labelled antibodies bound to said protein or peptide is the second signal according to the teaching of the present invention.

In one embodiment, a dataset for all wells of the chip is generated, and the data points associated with wells not containing exactly one cell, or not containing at least one cell, are removed. In another embodiment, a dataset for all wells of the chip is generated, and only the data points associated with wells containing exactly one cell, or containing at least one cell, are saved in a new dataset.

According to any one of the embodiments of the present invention, the chip data comprises any data and any measurement made in the chip. According to some embodiments, the chip data comprises the occupancy information of the wells. According to another embodiment, the chip data comprises the signal or the information obtained from Western blotting, including quantities and/or qualitative date about the measured protein(s) and/or peptide(s). According to some embodiments, the chip data comprises the dataset obtained from Western blot from all wells of the chip. According to another embodiment, the chip data comprises the dataset obtained Western blot from wells comprising only one cell. The wells comprising one cells are detected by the methods of the present invention.

According to one embodiment, the present invention provides a method of analyzing a single cell Western blot (scWestem) chip, the method comprising: a. preparing a cell suspension comprising from 50,000 to 300,000 cells/ml; b. seeding cells of the cell suspension into the wells of the scWestem chip; c. lysing the seeded cells; d. electrophoretically separating cell content on a gel; e. staining the DNA content in each well of the scWestem chip; f. measuring a fluorescent signal obtained from DNA staining to obtain a first signal comprising DNA data for each well of the scWestem chip; g. immobilized proteins and/or peptides in the gel or chip; h. staining the proteins and/or peptides and measuring a fluorescent signal obtaining from the staining of the proteins and/or peptides to receive the second signal; i. measuring the at least one peptide or protein; j. for each well of the scWestem chip, comparing the DNA data of the respective first signal to at least one predetermined threshold; k. for each well of the scWestem chip, determining, responsive to an outcome of the respective comparison, occupancy information of the respective well; and

1 responsive to the determined occupancy information for each well of the scWestem chip, outputting chip data of the scWestem chip, wherein the chip data comprises at least one of the (i) occupancy information of the respective well obtained in step ; (ii) second signal for each well; (iii) second signal for all wells; (iv) measurement obtained at step (f) for each well; (v) dataset comprising the results of the measurement for all wells comprising cells; and (vi) dataset comprising the results of the measurement for wells comprising one cell as detected in step (k). The order of some steps may vary.

Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES Materials and Methods

Human Islets

Human islets were provided by the Southern California Islet Cell Resources Center (SC-ICRC) at the City of Hope (Duarte, CA), and were approved for research by the City of Hope Institutional Review Board, with the written informed consent from each organ donor family.. Human islets were received 2-4 days after isolation and were dissociated as follows. Islets were transferred into a 50 ml conical tube and treated with 0.01 mg/ml DNase I (Sigma, St. Louis, MO) for 6 minutes in 37°C. Islets were then washed in Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, Waltham, MA) and treated with 0.25% trypsin- ethylenediamine tetra- acetic acid (EDTA) (Thermo Fisher Scientific, Waltham, MA) for up to 15 minutes in 37°C. Trypsinization was stopped by adding RPMI medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA), 100 U/ml penicillin and 100 pg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA). The cells were then passed through a 40-pm cell trainer (Thermo Fisher Scientific, Waltham, MA) and counted.

Single-cell Western Blot

Single islet cells were washed in Milo re-suspension buffer (ProteinSimple, San Jose, CA, USA) and diluted to a concentration of 1.0 x 10⁵ cells/ml. Milo chips were incubated for 10 minutes in a Petri dish with re-suspension buffer and 1 ml of single-cell sample was pipetted on top of each chip. After 5 minutes of incubation, well occupancy of approximately 15%-20% was determined by visualization under a light microscope and the chips were washed three times with re-suspension buffer. Each chip was then loaded into the Milo platform where cells were lysed with lysis buffer (ProteinSimple, San Jose, CA, USA) for 25 seconds, separated for 55 seconds at 240V and UV cross-linked for 4 minutes. The chips were then washed three times, 10 minutes each, with the Wash buffer (ProteinSimple, San Jose, CA, USA) in a new Petri dish. After washing, the chips were probed for 1-2 hours with primary antibodies diluted in the Antibody Dilution buffer (ProteinSimple, San Jose, CA, USA). Antibody concentrations were as follows: guinea pig anti-insulin (1:100, Dako A0564, Santa Clara, CA, USA), rabbit anti-somatostatin (1:50, Dako A0566, Santa Clara, CA, USA), mouse anti-glucagon (1:10, BD biosciences 565859, Franklin Lakes, NJ, USA) and rabbit anti-beta- actin (1:20, Cell- signaling 4967S, St. Louis, MO, USA). The chips were then washed three times with wash buffer in a new Petri dish and probed with secondary antibodies conjugated to Alexa fluorophores (488nm, 594nm and 647nm) or Cy3 (1:25, all from Jackson ImmunoResearch, West Grove, PA). Following secondary antibody incubation, the chips were washed three times, 15 minutes each, with wash buffer in a new Petri dish. The chips were then stained with 1 mg/ml DAPI (Santa Cruz Biotechnology, Dallas, TX, USA) for 5 minutes and washed three times, 5 minutes each, with wash buffer in a new Petri dish. The chips were then dried by centrifugation (2 minutes, 1000 x g) and scanned by either Innoscan 710 microchip scanner (Innopsys, Carbonne, France) or Keyence fluorescence microscope (Keyence, Osaka, Japan). The obtained .TIF images were analyzed by the Scout software (ProteinSimple, San Jose, CA, USA).

According to the manufacturer’s software, the fluorescence signal-to-noise ratio (SNR) is calculated at each discrete point in the lane based on the max signal divided by the root mean square (RMS) noise of the baseline. To ensure accuracy on the data of AUCs generated by the software, band shape and migration distance from the center of the well were manually reviewed to ensure that the fluorescence signal is unique to the specific, expected wavelength.

Immunofluorescence

Paraffin sections of pancreas from 4 donors were de-paraffinized in xylene followed by re hydration in ethanol. Antigen retrieval was done using Antigen Unmasking Solution (Vector laboratories, Burlingame, CA). Samples were blocked for 20 minutes at room temperature in blocking buffer containing 1% bovine serum albumin (BSA), 10% fetal donkey serum, and 0.2% saponin (Sigma, St. Louis, MO), and incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Antibody concentrations were as follows: guinea pig anti-insulin (1:300, Dako A0564, Santa Clara, CA, USA), rabbit anti-somatostatin (1:200, Dako A0566, Santa Clara, CA, USA), mouse anti-glucagon (1:2000, Sigma G2654, St. Louis, MO, USA). Slides were washed in PBS with 0.1% Tween (Sigma, St. Louis, MO) and incubated with secondary antibodies conjugated to Alexa fluorophores (488nm, 594nm and 647nm) (1:100, Jackson ImmunoResearch, West Grove, PA). DNA was stained with DAPI (Santa Cruz Biotechnology, Dallas, TX). The slides were mounted with Fluorescent Mounting Medium (Dako, Santa Clara, CA). Images were visualized under a ZEISS inverted LSM 700 microscope and processed using ZEN lite digital imaging software (Carl Zeiss, Oberkochen, Germany).

Insulin Dot-Blot assay

Recombinant human insulin (Mercodia, Uppsala, Sweden) was diluted in Milo antibody diluent (ProteinS imple) to 1 m unit per mΐ. The sample was then serially diluted by 10-fold over 4 orders of magnitude. Two 5 mΐ-spots from each concentration was gently placed on Milo chips, followed by incubation overnight at RT. The following day, the chips were processed, as described in the single-cell Western blot analysis above. To quantify the fluorescence signal in each spot, the images were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Statistical analysis was performed on Prism v.7.02 using 2-Way ANOVA corrected with statistical hypothesis testing. REML analysis was performed on the computer program JMP (SAS Institute, Cary, NC, USA).

Example 1. Identification of well occupancy with single versus double cells using a DNA dye.

A scWestem chip used in this study contains a total of 6,400 wells divided into 16 blocks of 400 wells each. A single-cell suspension was prepared by dissociating at least 1,000 islets per experiment with trypsin. To minimize the occurrence of multiple cells settling in one well, cells were diluted to a low density and the cell incubation time on the chip was limited to approximately 5 min. This strategy allowed the cells to settle in some (ranged 650-1,450 cells per 6,400 wells) but not all wells of the chip. Additionally, because the average diameter of endocrine cells is about 10 m the present disclosure used the chips specified with wells of 20 m in diameter, making doublets less likely to occupy a single well.

The present disclosure first addressed the issue of well occupancy. Under a light microscopy, the number of occupied wells and the number of cells in each well were counted manually. Doublets were detected in a small percentage (2-5%) of the occupied wells (not shown). Subsequently, cells were lysed in situ , and the protein contents were run by electrophoresis and crossed-linked to the chip.

The present examples first tested the utility of b-Actin, a housekeeping protein, as a proxy to detect cell doublets. The chips were probed with anti-P-Actin antibodies and fluorescence signals were detected in wells that were occupied with cells (Fig. 3A). The fluorescence signal in each well, expressed as the signal-to-noise ratio (SNR) along the migration distance, was determined and plotted by the manufacturer’s software, and was shown as a curve (Fig. 3B). Subsequently, the areas under the curve (AUCs) above the background (Fig. 3B, shaded area) were calculated, which incorporated the curve height and width into account and therefore represented the expression levels of b-Actin. Finally, the number of cells expressing specific ranges of b-Actin levels were made into a histogram (Fig. 3C). Differences in the expression levels of b-Actin in single cells varied almost 1,000 fold (Fig. 3C). When trying to differentiate between 1-cell and 2-cells occupancy, one can expect 2-fold difference in the presence/expression of a particular marker. However, considering that the distribution of b- Actin expression between the cells is much higher than that, b-Actin is not a reliable marker to detect doublets.

Since cell lysis is executed in situ on the chips, the present disclosure next examined whether the amount of DNA retained in the wells may be used to detect doublets. With the chips used in Fig. 3A, DNA was stained using the dye DAPI, which was detected around the well opening (see Fig. 3D). In the next experiments, live cells were loaded onto the chips, brightfield images taken, lysed and stained with the DNA dye. The images at the well openings showed that a well occupied by a single or double cell had one or two streaks, respectively (Fig. 3D), suggesting a potential correlation of DNA signals with doublets’ presence.

To further confirm, fluorescent signals from DNA staining were plotted along the positions of the wells and expressed as the values obtained by multiplying the AUC with the fluorescence SNR of each well (to augment signals for visualization). Two populations emerged: wells with higher (Fig. 3E, rectanges) and lower (Fig. 3E, circles) DNA contents. The wells were then individually and manually checked and confirmed to contain doublets or singles in the higher- and lower-signal wells, respectively (not shown). The AUCs from single- and double-cell occupied wells were further analyzed (Fig. 3F - squares lower DNA content, circles - higher DNA content), which showed a significant difference among the two groups. As described above, an SNR function was determined, the SNR function being a convolution of: a signal to noise ratio (SNR) signal of the fluorescent counts in relation to a distance from the center of the respective well; and a predetermined canonical peak shape. Thus AUC is thus the area under the curve of the SNR function between the two center local minimums. The AUC was then multiplied with the value of the width of the curve, i.e. the distance between the two local minimums, and the outcome of the multiplication was the analyzed value.

A clear emerged threshold allows clearly separating wells containing one cell and those containing 2 cells. Importantly, the average values of DNA signals from the two groups differed by approximately 2-fold. It is clear that using different DNA dye will result in different fluorescent values, however, in each case a new threshold will emerge allowing separating single-cell and double-cell wells. These results demonstrate that DNA staining can be used to detect wells with double occupancy.

Example 2. Detecting variable levels of peptide hormones in individual islet cells Pancreatic cell types

The islets of Langerhans are comprised of several different cell types: the most abundant being the insulin-secreting beta-cells followed by the glucagon-secreting alpha-cells and the somatostatin-producing delta-cells. The percentage of each type varies greatly between individuals. It had been generally believed that each sub-population of islet cells was homogeneous; however, recent evidence suggests beta-cell heterogeneity pertaining to gene expression levels and even function as shown by differences in glucose-responsive insulin secretion in different beta-cell subtypes. In order to truly understand the heterogeneity of the various cells in the islets, it is essential to investigate cells at the single-cell level. In recent years, there has been tremendous progress in the understanding of single islet cells by employing technologies which are restricted in certain aspects, such as difficulties in distinguishing cell doublets from single cells in RNA-seq or re-analyzing cells with new targets of interest with mass.

Recent evidence demonstrates the existence of immature beta-cells in normal adult mice. These immature “virgin” beta-cells were shown to trans-differentiate from alpha cells in the islets’ periphery and were able to differentiate into mature functioning beta-cells. The ability of islet cells to trans-differentiate from one endocrine cell type into another through a double-hormonal phase was shown under different. However, most studies on single islet cells disregard data containing double-hormones, assuming that they result from cell doublets . To date, whether human islets isolated from non-diabetic donors without apparent diseases contain double- hormonal cells remained unclear.

For these reasons, the present inventor tested a single-cell Western blot (scWestem) platform (Milo™) to detect in individual cells intracellular peptide hormones, which had never been demonstrated before. The present disclosure determined the cellular composition of isolated human islets from cadaveric donors and quantified the expression levels of peptide hormones in single cells. As exemplified hereinafter, it was possible to demonstrate in the non-diseased adult human islets the heterogeneity of hormone-expressing cells and the existence of double- hormonal cells that simultaneously express insulin and somatostatin or insulin and glucagon. These results suggest that trans- or de-differentiation could be a normal process in the islets of adult humans.

Mature peptide hormones typically have size less than 5K Daltons and unstable. Traditional Western blotting techniques often fail to identify peptides of such size range. In addition, hormones are stored in small vesicles in endocrine cells, making them less accessible to lysis detergents. To test the utility of scWestem for detecting intracellular peptide hormones, islets were dissociated, and single cells were loaded onto chips. To allow full extraction of the hormones from their vesicles and provide optimum separation, the present disclosure first determined the lysis and the migration time that succeeded for all three hormones under study: insulin (INS), somatostatin (SST) and glucagon (GCG) (see Materials and Methods). Subsequently, the optimized concentrations of antibodies for these 3 hormones were determined (as summarized below in Materials and Methods).

To determine the expression intensities of the three hormones among single cells, dissociated islet cells were loaded on chips, lysed, probed with primary antibodies of different species origins against INS, SST and GCG, followed by staining with various fluorescence-labeled secondary antibodies. The scheme of the experiment is presented in Fig. 4A. The fluorescence images for INS, GCG and SST were scanned, and subsequently, the chips were stained with DNA dye and the double-occupancy wells were excluded from the analysis according to the method of Example 1. Again, the fluorescence signals, expressed as SNR, in each well along the migration distance were determined and plotted. Each hormone was detected with a unique shape and expression levels of each hormone in each cell were determined by AUCs (Fig. 4B- D). The distance of the centers of the migration peaks for individual curves were recorded along the wells (not shown), which showed relatively consistent running distances for insulin and glucagon. The peaks for somatostatin, however, had a larger variance; the significance of this finding is unclear, but it is speculated that this could be due to the isoforms of somatostatin. Next, the expression levels of each hormone in each cell were and made into a histogram (Fig. 2E-G)). Differences as big as 70 fold in the protein expression levels of all 3 hormones were detected among single cells. Overall, these results demonstrate heterogeneity among individual islet cells expressing these 3 hormones.

Validation of quantitative data.

To rule out the possibility that the high variability of hormone expression in single cells shown in Fig. 4E-G was due to chip irregularities, three separate chips loaded with cells from a single donor were examined the number of insulin-expressing cells among the total population. It was found that the percent of insulin-positive cells was similar across the 3 chips: 17.1%, 19.3% and 19.4% . These numbers fell within the calculated 95% confidence interval between 17.1% to 20.1%, suggesting reproducibility of the chips.

Next, it was necessary to test whether the spatial positions on a single chip contributed to the variability in hormone signal detections. To evaluate the relationship between the spatial locations (by well numbers) and the variance of measured levels of insulin or glucagon, a variance-component estimation analysis with the restricted maximum likelihood (REML) statistics method was used. Spatial localization contributed about 0.5% to the variance while other factors (accounted for more than 99% of the variance in expression levels for both INS and GCG. Together, these results suggest that intra-chip locations, which are proxies for scanner scanning and antibody probing, did not affect the variance of hormone signals.

To further determine the amounts of INS that could be detected in a linear range, increasing amounts of recombinant human insulin were spotted on a chip as dots and probed with the primary antibody against INS followed by detection with fluorescence-labeled secondary antibodies (data not shown). A positive correlation was found between fluorescence signals emitted and the amount of recombinant human insulin ranged from 0.005 to 50 microunits per dot, with a correlation coefficient approaching 1 (R² = 0.9618). It is estimated that a human beta cell contains approximately 0.1 to 1.36 micro units of insulin. This result demonstrates that insulin amounts with a 10,000-fold difference encompassing the estimated values of insulin content per human beta cell can be measured on the scWestem chips. Example 3. Islet-cell composition analyses are comparable between scWestern and immunofluorescence staining

Traditional islet-cell composition studies have been performed using immunohistochemical staining on pancreatic tissue sections. Next, it was determined whether scWestern blot was comparable to the conventional method in assessing the fraction of hormone-expressing cells in islets. A total islets of 4 donors were analyzed. A region of the donor pancreas was removed, fixed, embedded in paraffin, sectioned, prepped for immunofluorescence staining (IF) for the 3 hormones: insulin (INS), somatostatin (SST) and glucagon (GCG) and the percent of hormone positive cells was determined. Subsequently, islets were isolated from the same donor pancreas, dissociated into single cells, ran on a chip and probed with the 3 antibodies. The total analyzed number of single cells (after elimination of double-occupancy wells according to method of Example 1) were between 1,565 and 2,427 single cells per donor. The results are presented in Table 1.

Table. 1 Comparison of scWestern blot and immunofluorescence staining (IF) analysis

The percent of insulin, glucagon, and somatostatin-expressing cells among the total islet cells were comparable between the two methods, demonstrating a high concordance between the scWestern and IF results in determining the proportions of specific hormone-expressing cells in islets. The observed present of hormonal cells are consistent to a prior reports, which showed that human beta- and alpha-cells range 25-80% (Mean 48.6%, Median 42.6%) and 5-70% (Mean 37.1%, Median 41%), respectively, in non-diabetic donor islets. However, it was noted that a significant higher percentage of cells did not express any of the 3 hormones in the scWestern method, which may be due to non-islet cells collected together with the islets during the isolation procedure. Regardless, these results demonstrate that scWestern is comparable to IF studies in determining the cellular composition of endocrine cells in islets.

Example 4. Single cells expressing double hormones are detected in islets from nondiabetic donors. It was tested whether single cells that express more than one hormone can be detected in islets from non-diabetic donors. Human islets, prepared from 4 donors, were dissociated, ran on chips and triple-stained with antibodies against INS, SST and GCG, as well as DNA staining. Any well with double-cell occupancy determined by the DNA staining was excluded from the analysis according to the method of Example 1. The results are presented in Table 2.

Table 2.

An average of 5.1% (n=35 out of 693) of GCG⁺ cells expressed INS and 12.5% (n=32 out of 252) of SST⁺ cells expressed INS, among 4 donors. No cell expressed both SST and GCG (not shown). These results unequivocally demonstrate the existence, at the protein level, of double- hormonal cells in non-diabetic donors.

Expression levels of hormones in individual cells were next analyzed. In the INS⁺SST⁺ double positive cells, somatostatin levels were significantly lower than that in INS SST⁺ single positive cells (Fig. 5, first 2 columns; FC=0.75, n=32, p<0.05). A lower level was also found for glucagon expression in INS⁺GCG⁺ double-positive cells (Fig. 5, middle 2 columns; FC=0.46, n=33, p O.OOl,). In contrast, there was no significant difference in insulin expression levels between double (INS⁺SST⁺ and INS⁺GCG⁺) and single-positive INS⁺GCG SST cells (Fig. 5, last 2 columns; FC=1.1, p=0.3 for both INS+GCG+ and INS+SST+). These results demonstrate that insulin is a dominant hormone expressed at higher levels in the double- hormonal cells.

Conclusions

In the examples provided above, scWestern was employed to detect and quantify intracellular peptide hormones and analyzed single islet cells. This platform has been shown to be a powerful tool in studying cancer and neuroscience, but its utility had not been shown in islet research, presumably due to the difficulties in analyzing small peptide hormones. Although single-cell mass cytometry can also measure levels of protein expression accurately in a single cell, it does not allow analysis of the same cells at a later time to gain further biological insights. In contrast, scWestern has the advantage of striping and re-probing with different antibodies on the same cells on a chip multiple time, as demonstrated previously.

However, implementation of scWestern technology could not be used to identify double- hormonal cells until the present invention. In scWestern technology some of the well may be occupied by two cells instead of one. Inability to unambiguously distinguish between wells comprising one and two cells prevented such research. A major new technical finding of the present invention is the effectiveness of DNA staining in detecting wells with single- versus double-cell occupancies (Fig. 3).

In terms of resolving the endocrine-cell composition within the islets, the examples provide above show that results obtained by scWestern were comparable to the standard IF analysis using pancreas tissue sections. Importantly, it was possible to measure levels of recombinant insulin encompassing 4 orders of magnitudes using scWestern (data not shows). Such a range of detection is difficult to achieve in IF analysis due to the commonly-used fluorescence molecules having overlapping emission wavelengths detectable by the microscope from the adjacent cells, which raise the background noise. Another significant finding of the examples provided was the higher abundance of other cells that did not express the three major hormones detected by the scWestern compared to the IF analysis. This could be due to the fact that IF analysis of islet composition is restricted to cells within the islet architectures, as recognized on the images by the spatial cell arrangement apart from the exocrine tissue. In contrast, from the routine islet isolation, the end products contain not only islets but also some non-islet cells, with the purity of islets varying between 60%-90% among donors. Therefore, scWestern provides a more accurate evaluation of cells beyond the endocrine cells, which may potentially affect transplantation outcomes. As mentioned above, scWestern allows the quantification of proteins in the same individual cells re-probed with new antibodies repeatedly, unlike previously-employed methods for islet composition analysis.

Beta-cell heterogeneity has been demonstrated by single-cell mRNA expression analysis in several studies. However, these studies did not validate insulin expression at the protein level. The current results presented herein not only confirmed the notion of heterogeneity among beta cells, but also provide new evidence that alpha and delta cells are heterogeneous as well. While alpha and delta cell heterogeneity has been implicated previously, the present study is the first to demonstrate the heterogeneity of human delta cells. A significant finding of this study is the discovery of insulin-positive cells that simultaneously express either somatostatin or glucagon, in isolated islets from normal human donors without apparent diseases. The existence of double-hormonal cells in human islets has been debated for many years and was associated with islet dysfunction and type 2 diabetes. Although not diagnosed with diabetes at the time of death, Donors 2 and 3, who showed higher percentages of double-hormonal cells compared to the other donors in the scWestem analysis (Fig. 5C), had higher levels of HbAlc (5.8%), indicating a pre-diabetic state. Although more donor samples are required to further confirm, this observation is consistent to the idea that stressed/dysfunctional islets are prone to de-differentiation. In the remainder normal donor samples, it was possible to detect single cells expressing two mature hormones at once (Fig. 5C), suggesting that de-differentiate or trans-differentiate may occur in healthy individuals.

While there are, on average, significantly more glucagon-positive cells than somatostatin-positive cells, the abundance of INS⁺SST⁺ double-positive cells (12.7%) was higher than that observed for INS⁺GCG⁺ double-hormonal cells (5.1%) (Fig. 5C). These results suggest that beta cells may de-differentiate by preferentially gaining the expression of SST than GCG.

Unexpectedly, the double-positive cells expressed lower levels of either somatostatin or glucagon while the levels of insulin were high and comparable to those measured in cells that express insulin alone. It is possible to speculate that beta cells from normal donors are generally healthy and de-differentiation may occur occasionally in a sub-population of beta cells that experience some levels of stress, which may trigger upregulation of SST or GCG while the expression of INS is maintained.

In summary, these example demonstrate here the utility of scWestem in detecting small peptide hormones and in studying heterogeneity of human islet cells. This finding could be made only after assuring that the analysis does not include wells comprising 2 cells. If two cells are present in the same well, it is impossible to establish whether the existence of two different hormones is due to heterogeneity of cells or due to the presence of two different cells each expressing different hormone. Only after elimination of doubles from the analysis it was possible to prove the heterogeneity, i.e. that one cell express to different hormones. The present scWestem method should also have a wide application to study hormone-producing cells of many other complex organs.

Example 5 In this experiment, a Milo chip was loaded using the procedure similar to that described in Materials and Methods to approximately 50% of its capacity. Cells were washed in Milo re suspension buffer (ProteinSimple, San Jose, CA, USA) and diluted to a concentration of 2.0 x 10⁵ cells/ml and loaded 7 minutes. The cells were lysed and separated on the chip and then stained with TOTOl (diluted 1:50,000) for 5 minutes with agitation. The chip was then imaged and for each well a DNA area under the curve was calculated and then multiplied by the width of the signal.

The median value was then calculated for local windows (40 wells per window) and a threshold was determined as 1.9 fold over median (black line in the graph). Wells with DNA AUCxWidth values over 1.9 fold above the local median were determined to be occupied by doublets (n=281) and the analysis continues with only single cells (n=2134).

In this experiment we succeeded to increase the number of loaded and effectively measured cells (i.e. signal from only single cells was considered) from about 600 to more than 2000 cells. In this way we collected much more information than in the past. Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

1. A method of analyzing a single cell Western blot (scWestern) chip, the method comprising: a. preparing a cell suspension; b. seeding cells of the cell suspension into the wells of the scWestern chip; c. lysing the seeded cells; d. electrophoretically separating cell content on a gel; e. staining the DNA content in each well of the scWestern chip; f. for each well of the scWestern chip, receiving a respective first signal comprising DNA data; g. for each well of the scWestern chip, comparing the DNA data of the respective first signal to at least one predetermined threshold; h. for each well of the scWestern chip, determining, responsive to an outcome of the respective comparison, occupancy information of the respective well; and i. responsive to the determined occupancy information for each well of the scWestern chip, outputting chip data of the scWestern chip.

2. The method according to claim 1, wherein the DNA staining is effected using a fluorescent labelling.

3. The method according to claim 2, wherein the fluorescent label of DNA is selected from DAPI, TOTO-1 iodide, Hoechst, 7-ADD, Crystal violet, and Ethidium bromide.

4. The method according to claim 2 or 3, wherein the respective DNA data for each well of the scWestern chip comprises a predetermined function of the fluorescence values from the respective well associated with the DNA staining.

5. The method according to claim 4, wherein the predetermined function comprises a multiplication of: an area under the curve (AUC) of the outcome of a convolution of a signal to noise ratio (SNR) signal and a predetermined peak shape, the SNR signal being of fluorescent counts in relation to a distance from the center of the respective well; and a width of the outcome of the convolution.

6. The method according to any one of claims 1 to 5, wherein the at least one predetermined threshold comprises a predetermined statistical attribute of the DNA data of the wells of the scWestem chip.

The method according to any one of claims 1 to 5, wherein the at least one predetermined threshold comprises a plurality of predetermined statistical attributes, each predetermined statistical attribute associated with a respective one of a plurality of sets of adjacent wells of the scWestem chip.

8. The method according to claim 6 or 7, wherein the predetermined statistical attribute comprises a median value.

9. The method according to any one of claims 1 to 5, wherein the at least one predetermined threshold comprises the lowest value of the DNA data of the wells of the scWestem chip.

10. The method according to any one of claims 1 to 5, wherein the at least one predetermined threshold comprises the lowest value of the DNA data of each of a plurality of sets of adjacent wells of the scWestem chip.

11. The method according to any one of claims 1 to 10, wherein, for each of the wells of the scWestem chip, the method further comprises, responsive to an outcome of the comparison to the at least one predetermined threshold, determining whether the respective well is occupied by more than one cell.

12. The method according to any one of claims 1 to 10, wherein, for each of the wells of the scWestem chip, the method further comprises, responsive to an outcome of the comparison to the at least one predetermined threshold, determining whether the respective well is occupied by at least one cell.

13. The method according to any one of claims 1 to 10, wherein the at least one predetermined threshold comprises at least one first predetermined threshold and at least one second predetermined threshold, and wherein, for each of the wells of the scWestem chip, the method further comprises: determining, responsive to an outcome of the comparison to the at least one first predetermined threshold, whether the respective well is occupied by more than one cell; and determining, responsive to an outcome of the comparison to the at least one second predetermined threshold, whether the respective well is occupied by at least one cell.

14. The method of claim 13, wherein the comparison to the first predetermined threshold is performed only for cells of the scWestern chip determined to be occupied by at least one cell.

15. The method of any one of claims 12 to 14, further comprising, for each of the wells of the scWestern chip determined to be occupied by at least one cell, subtracting from the

DNA data of the respective well a respective value associated with the DNA data of at least one well determined to not be occupied by at least one cell.

16. The method of claim 15, wherein the at least one well not occupied by at least one cell comprises the two of the wells closest to the respective well.

17. The method according to any one of claims 1 to 16, wherein the cell suspension comprises up to 800,000 cells/ml

18. The method according to claim 17, wherein the cell suspension comprises from 10,000 to 500,000 cells/ml.

19. The method according to any one of claims 1 to 18, wherein the chip data comprises the occupancy information of the wells of the scWestern chip.

20. The method according to any one of claims 1 to 19, further comprising immobilizing the proteins, peptides and/or polypeptides in the gel.

21. The method according to claim 20, further comprising staining the immobilized proteins and/or peptides.

22. The method according to claim 21, further comprising receiving a second signal obtained from the staining.

23. The method according to claim 22, further comprising, for each of the wells of the scWestern chip responsive to the received second signal, measuring at least one peptide or protein in the respective well.

24. The method according to claim 23, wherein measuring at least one peptide or protein comprises qualitative and quantitative measurement.

25. The method according to any one of claims 22 to 24, wherein the output chip data comprises the second signal or the results of the measurement of the at least one peptide or protein.

26. The method of any one of claims 22 to 25, further comprising, responsive to the occupancy information of each of the wells of the scWestern chip, generating a dataset comprising the results of the measurement for a subset of the wells.

27. The method of claim 26, wherein the subset of the wells excludes (i) wells that are not occupied by at least one cell; (ii) wells that are occupied by more than one cell; or (iii) both (i) and (ii).

28. The method of claim 26 or 27, wherein the chip data comprises the generated dataset.