CN113063646A - Cell fixing agent, cell fixing method and application - Google Patents
Cell fixing agent, cell fixing method and application Download PDFInfo
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- CN113063646A CN113063646A CN202110331046.1A CN202110331046A CN113063646A CN 113063646 A CN113063646 A CN 113063646A CN 202110331046 A CN202110331046 A CN 202110331046A CN 113063646 A CN113063646 A CN 113063646A
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The application relates to the field of cell biology, and particularly discloses a cell fixing agent, a cell fixing method and application. The cell fixing agent has the advantages that the specificity of the effective components of the cell fixing agent is specific to specific organic groups on proteins, the specific organic groups on the proteins are crosslinked through forming covalent bonds, a spacer arm is arranged between two ends with chemical activity, the length of the spacer arm is not less than 0.5 nanometer, and the spacer arm can be selectively cut off. The cell fixation method of the present application comprises (1) diluting the cell fixative with PBS buffer; (2) the cell sample is added to the diluted cell fixative for incubation. The cell fixative of the application is applied to immunofluorescence technology.
Description
Technical Field
The application relates to the field of cell biology, in particular to a cell fixing agent, a cell fixing method and application.
Background
With the development of high throughput sequencing technologies, sequencing costs are greatly reduced, which has prompted the development of various omics technologies and widespread applications in biological and medical research. In recent years, single cell sequencing has become a new focus in biomedical research. Traditional gene sequencing targets a large number of cells, reveals a general gene expression regulation or disease mechanism in the cells, but ignores the heterogeneity among individual cells. The development and development of single cell sequencing technology can help researchers analyze heterogeneity of cell populations and reveal differences among cells in aspects of gene expression, gene variation and the like.
In particular, single cell sequencing refers to sequencing the genome or transcriptome of a cell at the single cell level, and can be divided into single cell genome sequencing (single cell genome sequencing) and single cell transcriptome sequencing (single-cell transcriptome sequencing). Single cell sequencing technology can therefore reveal the unique phenotype that a small number of cells have. Single cell genomic sequencing technology can identify heterogeneity of tumor cell gene mutations or copy number variations on a single cell scale. For example, before the embryo transplantation of the test-tube infant, the screening of genetic diseases can be carried out by applying a single-cell genome sequencing technology, and the embryo which does not carry pathogenic gene mutation or copy number variation is screened for transplantation, thereby avoiding birth defects. The single cell transcriptome sequencing technology can detect gene expression from the single cell level, thereby avoiding gene expression from being averaged when sequencing a large number of cell transcriptomes, being beneficial to disclosing the regulation and control characteristics of gene expression in cells and discovering new cell types.
Single cell sequencing in addition to detecting heterogeneity in a population of cells, it is often necessary to detect specific cell populations, which may often be labeled with monoclonal antibodies to protein markers. Namely, target cells are found through immunostaining aiming at protein markers, and then the target cells are subjected to further nucleic acid detection.
Meanwhile, in medical research, there is also a need to simultaneously detect cell antigen expression and sequence nucleic acids. Because of the need for storage and transportation of cell samples, and the need for intracellular protein detection, cells are first fixed to maintain the integrity and stability of the protein antigen structure, and therefore, there is a need to develop a cell fixation method that is compatible with both intracellular protein detection and single cell sequencing (including DNA sequencing and RNA sequencing). Besides maintaining the structure and conformation of the protein antigen, the immobilization method can effectively protect the integrity of nucleic acid and is compatible with the current single cell sequencing method.
Currently, organic solvents such as aldehyde reagents and alcohol reagents are commonly used as cell fixatives. The aldehyde reagents immobilize the cells by forming chemical crosslinks, i.e., crosslinks soluble proteins, cytoskeletons, and nucleic acids within the cells, where the terminal groups of the proteins that can form crosslinks include amino groups, thiol groups, guanidino groups, hydroxyl groups, and aromatic rings.
For example, the current chinese patent application No. 2018115877086 discloses "a method for fixing cells and a cell fixing reagent", which uses glyoxal fixing solution instead of paraformaldehyde, and is less harmful to human health.
As another example, Chinese patent application No. 2019108971410 discloses a cell fixing method, which comprises fixing cells with 0.5-5 vol% formaldehyde solution, preferably 1-2 vol% formaldehyde solution, and more preferably 1.33 vol% formaldehyde solution, without special instrument for treating cells, and has low cost and simple operation.
Although the methylene bridge introduced by the aldehyde fixative fixed cells can well maintain the cell morphology and has good immunofluorescence staining effect, the methylene bridge can change the structure of a protein antigen to cause false negative results because the methylene bridge can enable the protein and the nucleic acid to form a cross-linked structure, and meanwhile, the fixative can damage the nucleic acid to be not beneficial to single cell sequencing.
Alcohol fixatives can cause dehydration of cells, thereby coagulating soluble proteins within the cells. This method of fixing cells affects cell morphology and the effect of immunostaining, and is less used in immunofluorescence staining.
The commonly used single cell genome amplification reagents are random primer amplification (DOP-PCR), Multiple Displacement Amplification (MDA) and multiple annealing circular cycle amplification (MALBAC). DOP-PCR is based on random combination of primers and genome, genome segments are amplified through PCR, but the genome amplification is prone to have bias due to PCR amplification, so the coverage of the whole genome amplification product is often less than 10%, and meanwhile, the base mismatching rate of PCR amplification reaction is high, the single cell genome amplification technology is not suitable for detection of single base mutation of tumor cells, the false positive rate is high, and the technology is less used along with the development of the technology.
The phi29DNA polymerase used by the MDA technology has strong DNA synthesis activity and nucleic acid chain replacement activity and high fidelity, so the method has the characteristic of high genome amplification coverage and is more suitable for detecting single-base mutation of tumor single cells. However, phi29DNA polymerase has low amplification efficiency on incomplete DNA, so that the method is only suitable for fresh cell samples and is not suitable for cell samples fixed by the traditional method. The MALBAC technology can be simultaneously used for single cell genome amplification of a fresh single cell sample and a fixed single cell sample, but the fidelity of the used DNA polymerase is not as high as that of phi29DNA polymerase, so that the fidelity is intermediate when detecting single base mutation.
The aldehyde fixed cell sample cannot use MDA for genome amplification due to certain damage of nucleic acid, while the DOP-PCR and MALBAC techniques can be used for nucleic acid amplification after damage, and are not suitable for detection of tumor base mutation due to insufficient fidelity. RNA in cells is usually in a single-stranded form, cell samples are fixed by aldehydes, and the RNA is easily damaged and broken, even degraded and not beneficial to detection.
Although the alcohol-fixed cells have good nucleic acid integrity and meet the experimental requirements of MDA amplification and RNA detection, the alcohol-fixed cells often have poor signals in immunostaining for protein antigens. The requirement of the novel medical examination on simultaneous detection of single cell sample protein and nucleic acid puts higher requirements on the use of a cell fixing agent, and the common cell fixing agent hardly considers protection of protein antigen and stabilization of intracellular nucleic acid.
Disclosure of Invention
In order to protect protein antigens and stabilize intracellular nucleic acids, the application provides a cell fixing agent, a cell fixing method and application.
In a first aspect, the present application provides a cell fixative, which adopts the following technical scheme:
a cell fixative is prepared by cross-linking specific active components with specific organic groups on protein through covalent bond formation, and has a chemically active spacer arm between two ends, wherein the length of the spacer arm is not less than 0.5 nm, and the spacer arm can be selectively cut off.
By adopting the technical scheme, the protein can be specifically crosslinked, and meanwhile, the spacer arm is arranged, so that the activity of the antigen can be retained to the maximum extent, and the antigen can be conveniently identified and immunostained; meanwhile, the nucleic acid in the cells can be protected from loss due to the formation of a cross-linked structure. Single cell sequencing can be achieved by subsequent release of the nucleic acid molecule by cleavage of the spacer arm of the cross-linking molecule (e.g., dithiothreitol or other cleavage reagent).
Preferably, the effective component is amino or sulfhydryl groups on the crosslinked protein.
Preferably, the effective component is one or a combination of a homobifunctional cross-linking agent with amino reactivity and a heterobifunctional cross-linking agent with amino reactivity and a photoactivation azidobenzene compound.
Preferably, the active ingredient is one or a combination of two of 3,3' -dithiodipropionic acid bis (N-hydroxysuccinimide ester) and 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester.
By adopting the technical scheme, the 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) and the 3- (2-pyridyl-dithio) propionic acid N-hydroxysuccinimide ester are adopted as the effective components of the cell fixative. The 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) and the 3- (2-pyridyl-dithio) propionic acid N-hydroxysuccinimide ester can be specifically crosslinked aiming at organic groups (such as amino and sulfhydryl) on cell proteins, and both contain a spacer arm which can be selectively cut off, so that the activity of an antigen can be retained to the maximum extent, and the antigen can be conveniently identified and immunostained; meanwhile, the nucleic acid in the cells can be protected from loss due to the formation of a cross-linked structure.
In addition, the complete structure and conformation of the protein antigen can be maintained, and the cell permeability is increased, so that antibodies or other dye molecules can enter cells without additionally using a perforating agent to perforate a cell membrane of a fixed sample, and the method has an excellent nucleic acid stabilizing effect, can be compatible with various single-cell nucleic acid sequencing technologies, and can finish sequencing analysis of single-cell DNA or RNA.
Preferably, the cell fixative is prepared by dissolving bis (N-hydroxysuccinimide ester) 3,3 '-dithiodipropionate and N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate in a solvent to obtain a bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate solution and a N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate solution, respectively, and mixing the two solutions.
Preferably, the solvent is one of anhydrous dimethyl sulfoxide and N, N-dimethylformamide.
Preferably, the concentration of the 3,3' -dithiodipropionic acid bis (N-hydroxysuccinimide ester) solution and the concentration of the 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester solution are each 10 to 400 mM.
In a second aspect, the present application provides a method for cell fixation using the above cell fixative, comprising the steps of:
a cell fixing method using the cell fixing agent comprises the following steps:
(1) diluting the cell fixing agent by using a buffer solution;
(2) the cell sample is added to the diluted cell fixative for incubation.
By adopting the technical scheme, the structure and conformation of the protein antigen and the integrity of nucleic acid can be protected, so that the protein antigen can be used for simultaneously carrying out immunostaining of protein in cells and biomedical detection of single cell sequencing (DNA and RNA). In addition, the kit and the practical method thereof can be used for preserving cell samples, stabilizing and/or preparing compositions of nucleic acid in biological samples.
Preferably, in step (2), the cell sample is resuspended in the buffer to obtain a cell solution, and then the cell solution is added to the diluted cell fixative for incubation.
In a third aspect, the present application provides an application of the above cell fixative in immunofluorescence technology, which adopts the following technical scheme:
an application of the cell fixing agent in immunofluorescence technology.
In summary, the present application has the following beneficial effects:
1. the cell fixative uses 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) and 3- (2-pyridyl-dithio) propionic acid N-hydroxysuccinimide ester as effective components. The 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) and the 3- (2-pyridyl-dithio) propionic acid N-hydroxysuccinimide ester can be specifically crosslinked aiming at organic groups (such as amino and sulfhydryl) on cell proteins, and both contain a spacer arm which can be selectively cut off, so that the activity of an antigen can be retained to the maximum extent, and the antigen can be conveniently identified and immunostained; meanwhile, the nucleic acid in the cells can be protected from loss due to the formation of a cross-linked structure.
2. The method can maintain the complete structure and conformation of the protein antigen, and simultaneously increase the cell permeability, so that the antibody or other dye molecules can enter cells without additionally using a perforating agent to perforate the cell membrane of a fixed sample, and the method has excellent nucleic acid stabilizing effect, can be compatible with various single cell nucleic acid sequencing technologies, and further completes the sequencing analysis of single cell DNA or RNA.
Drawings
FIG. 1 is a photograph of the immunofluorescence staining of HK2 after fixation of cell line RT 4;
FIG. 2 is a gel electrophoresis image of the cell line RT4 single cell genome amplification DNAPR;
FIG. 3 is a graph showing the results of single cell genome-wide copy number variation detection after immobilization of cell line RT4 using bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate/N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate;
FIG. 4 is a diagram showing the result of detecting the whole genome copy number variation of RT4 live cell single cell;
FIG. 5 is a drawing of identification of male fetus sample sex chromosome STR typing, wherein A: STR typing (XY) of target cells; b: maternal cell STR typing (XX); c: villus STR typing (XY);
FIG. 6 is a capillary electrophoresis of amplified cDNA from the transcriptome of cell line RT4 single cell.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
In the application, the raw materials are all commercially available products.
Examples
Example 1
Example 1 discloses a cell fixative composition comprising bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate and N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionate as active ingredients. Wherein the 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) is homotype bifunctional cross-linking agent with amino reaction activity, and the molecular formula is C14H16O8N2S2The structural formula is as follows:
the 3- (2-pyridyl disulfide group) propionic acid N-hydroxysuccinimide ester is a heterobifunctional cross-linking agent with amino reaction activity and photoactivation of an azidobenzene compound, and the molecular formula of the heterobifunctional cross-linking agent is as follows: c12H12N2O4S2The structural formula is as follows:
dissolving the 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) and 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester powder in anhydrous dimethyl sulfoxide to prepare a 50mM 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) solution and a 50mM 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester solution, and mixing the 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester) solution and the 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester solution in equal volume to obtain a cell fixative for later use. When the mixture is stored, the 3,3' -dithiodipropionic acid bis (N-hydroxysuccinimide ester) solution and the 3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester solution can be frozen at-80 ℃.
Example 1 also discloses a cell fixation method comprising the steps of:
(1) diluting the cell fixing agent in example 1 by using PBS buffer solution with the pH value of 7.4;
(2) taking a tumor cell sample, firstly suspending the tumor cell sample in PBS buffer solution with the pH value of 7.4 to obtain tumor cell solution, then adding the tumor cell solution into the diluted cell fixing agent, and incubating for 40 minutes at room temperature.
The proportions of the components are shown in Table 1
Examples 2 to 3
The difference from example 1 is that the components of the cell fixative are different, and the details are shown in Table 1.
Table 1 ingredient distribution ratio table of example 1
Example 4
The difference from example 1 is that the concentration of the bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate solution and the concentration of the N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionate solution are both 10 mM.
Example 5
The difference from example 1 is that the concentration of the bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate solution and the concentration of the N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionate solution are both 400 mM.
Example 6
The difference from example 1 is that powders of bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate and N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionic acid were dissolved in N, N-dimethylformamide, respectively.
Comparative example 1
The difference from example 1 is that the effective component of the cell fixative was Polyformaldehyde (PFA), which was dissolved in anhydrous dimethyl sulfoxide to prepare a paraformaldehyde solution with a concentration of 50 mM.
Application example 1
An application of the cell fixative in the immunofluorescence technique, in particular to a monoclonal antibody of Hexokinase 2(HK2) for cell immunofluorescence staining, comprises the following steps,
a) the tumor cell sample fixed in the example 1 is dripped on an adhesive glass slide and is stood for 30 minutes at room temperature;
b) centrifuging and throwing the cells to the surface of the glass slide by using a piece throwing machine;
c) discarding the liquid, and washing the surface of the slide three times by using PBS buffer;
d) blocking with 2% fetal bovine serum-bovine serum albumin (2% FBS-BSA) solution for 1 hour at room temperature;
e) HK2 monoclonal antibody was expressed as 1: 100 was diluted to working concentration with PBS buffer. Discarding the blocking solution of the endocoat cell sample, adding the HK2 monoclonal antibody, and incubating for 8 hours at 4 ℃;
f) discarding the HK2 monoclonal antibody solution, and washing three times by using PBS buffer;
g) fluorescently labeled secondary antibodies were labeled according to 1: 400, diluting the mixture to a use concentration by using a PBS buffer solution, discarding a blocking solution, adding a diluted secondary antibody use solution, and incubating for 1 hour at room temperature;
h) discarding the secondary antibody solution, washing three times by using PBS buffer solution, adding 1 Xconcentration DAPI nuclear dye, and incubating for 10 minutes at room temperature;
i) washed three times with PBS buffer and fluorescence image acquisition was performed using a fluorescence microscope.
The results are shown in FIG. 1.
Application comparative example 1
The difference from application example 1 is that the cell fixative provided in comparative example 1 is used, and the test results are shown in fig. 1.
Application example 2
The cell fixative described in example 1 is used for fixing and immunofluorescence staining of tumor cell RT4 cell samples, and target cells are recovered for detecting single cell DNA whole genome copy number variation. The method specifically comprises the following steps:
a) using 0.25% pancreatin to digest the cultured tumor cells HK2, the tumor cells HK2 were resuspended in 1 ml of cell complete medium;
b) centrifuging the cell suspension to collect cell sediment under the centrifugation condition of 450g for 5 minutes at 4 ℃, washing the cell sediment twice by using PBS buffer solution, and finally suspending the cell sediment in 250 microliters of buffer solution;
c) tumor cells HK2 were pre-fixed according to the procedure in example 1;
d) the fixed tumor cells HK2 were subjected to immunofluorescence staining according to the procedure in application example 1;
e) recovering the HK2 positive single-cell sample by using a micromanipulation platform, and transferring the single-cell sample into 5 microliters of cell lysate;
f) incubating the single cell sample at 25 ℃ for 1 hour in the presence of dithiothreitol DTT to de-crosslink;
g) lysing single cells to release genomic DNA: at 55 ℃ for 3 hours; 15 minutes at 85 ℃;
h) single cell genomic DNA preamplification: uniformly mixing 30 microliters of pre-amplification buffer solution and 1 microliter of pre-amplification enzyme, and adding 30 microliters of reaction solution into the single cell sample; the reaction conditions were 94 ℃ for 3 minutes; 8 cycles (20 ℃, 40 seconds; 30 ℃, 40 seconds; 40 ℃, 30 seconds; 50 ℃, 30 seconds; 60 ℃, 30 seconds; 70 ℃, 4 minutes; 95 ℃, 20 seconds; 58 ℃, 10 seconds); i) single cell genomic DNA amplification: mixing 30 microliter of amplification buffer solution and 1 microliter of amplification enzyme uniformly, and adding 30 microliter of reaction solution into the single cell sample; the reaction conditions were 94 ℃ for 30 seconds; 17 cycles (94 ℃, 20 seconds; 58 ℃, 30 seconds; 72 ℃,3 minutes).
j) PCR assay using single cell genomic amplified DNA quality control primers: 95 ℃ for 3 minutes, 30 cycles (95 ℃, 30 seconds; 60 ℃, 30 seconds; 72 ℃, 30 seconds); 72 ℃ for 5 minutes.
k) Detecting the PCR quality control result by gel electrophoresis;
the results are shown in FIGS. 2-4.
Application example 3
The cell fixing agent in the embodiment 1 is used for fixing and immunofluorescence dyeing of a cervical scraping cell sample, and target cells are recovered for STR detection.
The cervical scrape cytological specimen is collected, and the pre-fixing and cellular immunofluorescent staining of the cervical scrape cytological specimen is completed according to the experimental procedures described in example 1. STR sequencing was further performed on rare HK2 positive cells in cervical scrape samples. The relevant experimental procedures were as follows:
a) collecting a cervical scraping mucus sample, and carrying out enzymolysis digestion to obtain a single cell suspension sample;
b) the single cell suspension sample is fixed by using the compound composition fixing agent, and the experimental operation method refers to example 1;
c) finishing immunofluorescence staining of the single cell suspension and finishing fluorescence image acquisition according to the experimental operation steps in the application example 1; d) recovering the HK2 positive single-cell sample by using a micromanipulation platform, and transferring the single-cell sample into 4 microliters of PBS buffer;
e) the compound composition fixing agent can be subjected to decrosslinking in the presence of DTT, 3 microliters of DTT cell lysate containing dithiothreitol is added into a single cell sample and incubated at 25 ℃ for 1 hour to decrosslink, and genomic DNA is released at 65 ℃ for 10 minutes;
f) taking out the sample, immediately placing the sample on ice, and adding 3 microliters of termination reaction solution;
g) single cell genomic DNA amplification: mixing 29 microliter of amplification buffer solution, 1 microliter of amplification enzyme and 10 microliter of molecular level water uniformly, and adding 40 microliter of reaction solution into the single cell sample; the reaction condition is 30 ℃ and 8 hours; 15 minutes at 65 ℃;
h) single cell genome amplification product quality control PCR experiment reference application example 2
i) Performing STR detection on the qualified single cell genome amplification product;
the results are shown in FIG. 5.
Application example 4
The cells described in example 1 are used for fixing and immunofluorescent staining of a tumor cell RT4 cell sample, and target cells are recovered for detecting the single-cell mRNA gene expression profile. The relevant experimental procedures were as follows:
a) the pre-immobilization of tumor cells refers to the related operation in example 1, the immunofluorescence staining experimental operation of HK2 refers to the related operation in application example 1, and RNase inhibitor is added into buffer;
b) recovering a HK2 positive single-cell sample by using a micromanipulation platform, transferring the single-cell sample into 4 microliters of cell lysate, wherein the main components of the cell lysate comprise Triton-X100, an RNase inhibitor, dithiothreitol DTT, dNTP, a primer and molecular water;
c) placing the single cell sample at 25 ℃ to incubate for 1 hour for de-crosslinking, taking out the single cell sample and placing the single cell sample on ice at 72 ℃ for 3 minutes;
d) single cell transcriptome mRNA pre-amplification: sequentially adding 6 microliters of reaction solution into a single cell sample, namely 2 microliters of reverse transcription buffer solution, 2 microliters of betaine, 0.5 microliters of MgCl2, 0.25 microliters of RNase inhibitor, 0.1 microliters of TSO joint, 0.5 microliters of reverse transcriptase and 0.65 microliters of molecular-grade water; the reaction conditions were 42 ℃ for 90 minutes; 10 cycles (50 ℃, 2 min; 42 ℃, 2 min), 70 ℃, 15 min;
e) single cell transcriptome cDNA amplification: mixing 12.5 microliters of PCR amplification buffer solution, 0.25 microliters of amplification primers and 2.25 microliters of molecular-level water uniformly, and adding 15 microliters of reaction solution into the single-cell sample; the reaction conditions were 98 ℃ for 3 minutes; 18 cycles (98 ℃, 20 seconds; 67 ℃, 15 seconds; 72 ℃, 6 minutes); 72 ℃ for 5 minutes.
f) The single cell transcriptome cDNA amplification products were mass-checked using capillary electrophoresis.
The results are shown in FIG. 6.
Analysis of detection results
Referring to fig. 1, after a 3,3' -dithiodipropionic acid bis (N-hydroxysuccinimide ester)/3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester compound composition is used as a tumor cell fixative to fix cells, an immunofluorescence staining experiment is carried out on a cell sample, which shows that the novel fixative can protect the structural integrity of cell protein antigens excellently, increase the cell permeability, and the immunofluorescence staining experiment effect of target protein is equivalent to that of the traditional immunofluorescence staining experiment after paraformaldehyde fixes cells.
Secondly, referring to fig. 2, after a cell line RT4 is fixed by a composition of bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate/N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate, the cell HK2 is subjected to immunofluorescence staining after the fixation according to example 1, a target cell unicell sample is recovered, MDA is used for unicell whole genome amplification after the decrosslinking, and PCR is performed on the amplified DNA by using quality control aiming at 22 chromosomes, so that the result shows that the novel fixative has excellent protection effect on the unicell DNA, the fixed unicell sample can still be subjected to unicell whole genome amplification by using the MDA technology, and the genome coverage of the amplification product is good.
Referring to fig. 3 and 4, after the 3,3' -dithiodipropionic acid di (N-hydroxysuccinimide ester)/3- (2-pyridyl-dithio) propionic acid N-hydroxysuccinimide ester compound composition is used for fixing cells, the integrity of the intracellular genome DNA can be stabilized, the tumor single cell amplified genome DNA can be recovered for detecting the chromosome copy number variation, and the result shows that the fixed cell sample is similar to the result of a living cell.
And fifthly, referring to fig. 5, the cell fixative provided by the application can be applied to STR detection.
Fourthly, referring to fig. 6, the cell fixative provided by the application has better protection effect on single cell RNA.
It should be noted that the detection results of the application examples 1-4 using the cell fixative obtained in example 1 are the same or similar to those of the application examples corresponding to the cell fixatives obtained in example 2-6, and are not repeated herein.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A cell fixative, characterized in that the effective component specifically aims at the specific organic group on the protein to crosslink through forming covalent bond, the middle of the two ends with chemical activity contains a spacing arm, the length of the spacing arm is not less than 0.5 nanometer, and the spacing arm can be selectively cut off.
2. The cell fixative according to claim 1, wherein the specific group on the protein to be cross-linked is an amino group or a thiol group.
3. The cell fixative as claimed in claim 1, wherein the active ingredient is a combination of one or more of a homobifunctional cross-linking agent having amino reactivity and a heterobifunctional cross-linking agent having amino reactivity and a photoactivated azidobenzene compound.
4. The cell fixative according to claim 1, wherein the active ingredient is a combination of one or both of bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate and N-hydroxysuccinimide ester of 3- (2-pyridyldithio) propionate.
5. The cell fixative according to claim 3, wherein the cell fixative is prepared by dissolving bis (N-hydroxysuccinimide ester) 3,3 '-dithiodipropionate and N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate in a solvent to obtain a bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate solution and a N-hydroxysuccinimide ester 3- (2-pyridyldithio) propionate solution, respectively, and mixing them.
6. The cell fixative according to claim 4, wherein the solvent is one of anhydrous dimethylsulfoxide and N, N-dimethylformamide.
7. The cell fixative according to claim 3, wherein the concentration of the bis (N-hydroxysuccinimide ester) 3,3' -dithiodipropionate solution and the N-hydroxysuccinimide ester solution of 3- (2-pyridyldithio) propionate are each 10 to 400 mM.
8. A method of cell fixation using the cell fixative of any one of claims 1-7, comprising the steps of:
(1) diluting the cell fixing agent by using a buffer solution;
(2) the cell sample is added to the diluted cell fixative for incubation.
9. The method according to claim 8, wherein in step (2), the cell sample is resuspended in the buffer solution to obtain a cell solution, and then the cell solution is added to the diluted cell fixative for incubation.
10. Use of the cell fixative of any one of claims 1-7 in immunofluorescence techniques.
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