CN111239028A - High-yield high-activity single cell sorting method - Google Patents
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Abstract
The invention discloses a single cell sorting method with high yield and high activity, the activity of the single cells obtained by sorting is higher, and the monoclonal formation rate of a 96-pore plate is as high as 95.8 percent; the single cells after sorting can form monoclonals with high efficiency, and more effective single cell gene data can be directly obtained through sorting; the activity of the sorted cell population completely meets the single cell sequencing requirement, and the bottleneck problem of multi-parameter screening of the cell population is solved. The single cell sorting method has higher single cell yield and higher cell activity, the high yield is beneficial to sorting rare cells and precious cells, the high activity can ensure the survival rate of the cells, and the problem of purifying multi-parameter specific target cell populations is solved.
Description
Technical Field
The invention relates to a single cell sorting method with high yield and high activity.
Background
The purification of target cell populations by flow cytometric sorting is currently the most common experimental method, especially for multiparameter defined cell populations, sorting being the best purification method. The purity, yield and activity are the main indexes for evaluating the sorted cells, and especially with the further development of single cell research, higher requirements are put on the sorting yield (pore plate sorting, especially rare cells) and activity.
Through reference of documents and experimental verification, factors influencing the sorting purity can be effectively controlled, so that the improvement of the sorting yield and the activity are particularly critical. Andrew Riddell et al invented a method for calculating the yield of target particles by detecting the number of target particles in the central liquid stream, and the calculated maximum yields were 76.41% and 82.68%, and there was still room for improvement. Under the condition of ensuring the highest yield, the activity of the sorted cells directly determines the proliferation condition of the cells and the formation rate of monoclone, and the most important thing is that the activity of the sorted cells can not meet the requirement of a single cell sequencer (85%), which has become a great obstacle in single cell sequencing experiments.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a single cell sorting method with high yield and high activity. The method adopts a flow cytometry sorting instrument to obtain single cells or cell populations, (1) the single cells of the sorted rare cells can efficiently form monoclonals, or more effective single cell gene data can be directly obtained through sorting; (2) the activity of the sorted cell population completely meets the single cell sequencing requirement, and the bottleneck problem of multi-parameter screening of the cell population is solved.
In order to achieve the purpose, the invention provides the following technical scheme:
a single cell sorting method with high yield and high activity comprises the following steps:
(1) jurkat cell culture: jurkat cells were cultured in RPMI-1640 medium containing 10% FBS by volume and 1% p/s by volume, at 37 ℃ and 5% CO by volume2Culturing in incubator every other dayChanging the liquid, discarding 4/5 volume of cell suspension when the cells grow to 80%, adding an equal volume of fresh culture medium, and carrying out cell passage by the method;
(2) jurkat cell staining: taking the jurkat cell suspension obtained in the step (1), centrifuging 300g to remove a supernatant, adding 0.5ml of fresh culture medium for resuspension, dyeing by using a fluorescent dye CFSE (color filtration dye), dyeing for 20 minutes at room temperature in a dark place, and then dyeing for 5 minutes at 37 ℃ in a Horchest33342 in a dark place; centrifuging to remove supernatant, resuspending in PBS buffer solution and filtering with a 40 μm cell sieve to obtain double-stained jurkat cells for sorting;
(3) setting single cell sorting conditions: calibration of drop delay: firstly, finding a liquid drop delay value by using an instrument automatic liquid drop delay calculation method, and then further correcting the liquid drop delay value by using a manual liquid drop delay regulation method until the liquid drop delay value completely meets the condition of the optimal liquid drop delay, namely, continuously taking 10 liquid drops under different liquid drop delay values, wherein the number of target particles in the fifth liquid drop is the most, only the fifth liquid drop has the target particles, determining the liquid drop delay value at the moment as the optimal liquid drop delay, and carrying out single cell sorting under the condition; and setting sorting conditions: a nozzle with the diameter of 100 mu m, the pressure of sheath fluid is 30psi, the pressure difference between sample pressure and sheath fluid is 0.3psi, the purity mode is selected to be single, and the drop envelope is selected to be 0.5; adjusting the optical path of the instrument, detecting QC, calculating the liquid drop delay according to the method, adjusting the sorter to an SD electroless sorting mode for sorting single cells of a 96-well plate and a 1536-well plate, pre-paving sheath liquid on the well plate by using the sorter, correcting the sorting position, then taking the cell suspension of the double-stained jurkat cells in the step (2) for sorting, and sorting 1 cell in each well of the 1536-well plate.
Further, single cell yield identification is also included: after the single cells are sorted to a 1536-well plate, each well of the 1536-well plate is scanned by blue light and green light of a multi-well plate cell imager respectively, imaging records are carried out, the cell condition in each well is confirmed by double fluorescence, when only one double-stained cell exists, one single cell is considered to be successfully sorted, the score is 1, the score is 0 for other wells, and the yield of the single cells is counted.
Further, single cell viability identification is also included: sorting the single cells to a 96-well plate, placing the plate in a carbon dioxide incubator for continuous culture, and observing the formation condition of the single cells; single cell viability was analyzed based on the single cell colony formation.
Further, in the single cell yield identification, a BioTek rotation 1 multi-well plate cell imager is adopted.
Further, in single cell viability assay, sorted cells were received in receiving medium RPMI-1640 medium plus 10% FBS by volume fraction.
The application of the high-yield high-activity single cells in single cell sequencing is realized, and the high-yield high-activity single cells are obtained by adopting the sorting method.
The invention has the beneficial effects that:
(1) the single cell sorting method adopts a flow cytometry sorter to obtain single cells or cell populations, can obtain the single cells with high yield and high activity, the yield of a 1536 pore plate is 53 percent, and the yield of a 96 pore plate (calculated by the number of formed monoclonals) is 91.6 percent; the single cells obtained by sorting have high activity, and the monoclonal formation rate of a 96-pore plate is up to 91.6%; the single cells after sorting can form monoclonals with high efficiency, and more effective single cell gene data can be directly obtained through sorting; the activity of the sorted cell population completely meets the single cell sequencing requirement, and the bottleneck problem of multi-parameter screening of the cell population is solved.
(2) The single cell sorting method has higher single cell yield and higher cell activity, the high yield is beneficial to sorting rare cells and precious cells, the high activity can ensure the survival rate of the cells, and the problem of purifying multi-parameter specific target cell populations is solved.
Drawings
FIG. 1 shows the yield of fluorescent microspheres under automatic droplet delay and under manual calibration of droplet delay.
FIG. 2 shows R-max values and purities of two populations of medium and high fluorescence intensity among CST fluorescent microspheres sorted by R-max method.
FIG. 3 is a statistical graph of the rate of drop in yield for the case of a drop delay deviating from 0.3 for four experimental conditions.
FIG. 4 is a graph showing the results of apoptosis experiments performed on different receiving solutions.
FIG. 5 is a graph showing the results of proliferation experiments on cells after sorting four different receiving solutions.
FIG. 6 is a graph of statistics of yield of 1536 well plate high throughput sorted single cells under optimized conditions.
FIG. 7 is a monoclonal image formed after sorting of single cells in a 96-well plate under optimized conditions.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention.
The cells, chemicals, reagents, instruments, etc. used in the following examples are commercially available.
The invention relates to a sorting method for obtaining high-yield and high-activity single cells by using a flow cytometry sorter, which comprises the following steps:
in a specific embodiment, the flow cytometer used was Astrios mofloEQ(BECKMANCOULTER)；
(1) Jurkat cell culture: jurkat cells (Jurkat cells commercially available from Atccmanassas Va, U.S. supplier; the single cell sorting method of the present invention is not limited to Jurkat cells, but the sorting method of the present invention can also be used for sorting other single cells) were cultured in RPMI-1640 medium containing 10% FBS by volume fraction and 1% p/s diabody (i.e., penilin-Streptomycin diabody), 37 ℃ and 5% CO by volume fraction2Culturing in an incubator, changing liquid every other day, carrying out passage for 3 days, discarding 4/5-volume cell suspension, adding fresh culture medium with the same volume, and carrying out cell passage by the method; the fresh culture medium was: RPMI-1640 medium with 10% FBS by volume fraction and 1% p/s double antibody by volume fraction, the method refers to: discarding 4/5 volumes of cell suspension and adding the same volume of fresh medium;
(2) jurkat cell staining: taking 5ml of the jurkat cell suspension obtained in the step (1), centrifuging at a speed of 300g to remove supernatant, adding 0.5ml of fresh culture medium (namely RPMI-1640 culture medium containing 10% of FBS by volume and 1% of p/s double antibody by volume), re-suspending, then staining with 1. mu.l of diluted fluorescent dye CFSE (specifically, diluting the fluorescent dye CFSE with PBS buffer solution with pH of 7.2-7.4, wherein the volume ratio of the CFSE to the PBS buffer solution is 1: 1000, and then staining is carried out after dilution, and the concentration of the fluorescent dye CFSE is 5. mu. mol/L after dilution), staining for 20 minutes at room temperature in the dark, and then staining for 5 minutes at 37 ℃ in the dark at Hoechst 33342 (100X) kit of Hoechst 33342 living cell staining solution of Biyunshi Biotech Limited, directly diluting by 100 times and then using the kit); centrifuging to remove supernatant, resuspending in PBS buffer (pH 7.2-7.4) and filtering with 40 μm cell sieve to obtain double-stained jurkat cells for sorting;
(3) setting single cell sorting conditions: calibration of drop delay (drop delay): in order to ensure the yield of single cells, the invention utilizes the manual regulation function to further regulate on the basis of automatically calculating the liquid drop delay by an instrument until the liquid drop delay completely meets the condition of the optimal liquid drop delay, the instrument defaults that the interval between different liquid drop delay values is 1 to obtain different liquid drop delay values, 1 liquid drop is taken under each liquid drop delay value, and 10 liquid drops are continuously taken; among the 10 sorted droplets, 50 fluorescent microspheres were present in the fifth droplet, and the number of fluorescent microspheres was 0 in the fourth and sixth droplets, and the droplet retardation at this time was considered to be the optimum value, that is, the droplet retardation value corresponding to the fifth droplet was the optimum droplet retardation value.
The instrument has the function of automatically calculating the delay value of the liquid drop, the default interval of different liquid drop delay values of the instrument is 1, 1 drop is taken under each liquid drop delay value, 10 drops are continuously taken, generally, the number of fluorescent microspheres in the fifth drop is the largest, two or more fluorescent microspheres exist in the fourth drop and the sixth drop, at the moment, the number of the fluorescent microspheres in the fourth drop and the sixth drop needs to be input into instrument software, the instrument can continuously calculate the liquid drop delay according to the number until 50 fluorescent microspheres exist in the fifth drop, no fluorescent microspheres exist in the fourth drop and the sixth drop, and the liquid drop delay value corresponding to the fifth drop is the optimal liquid drop delay value.
For example, the 10 different droplet retardation values are 27.6, 28.6, 29.6, 30.6, 31.6, 32.6, 33.6, 34.6, 35.6, and 36.6, respectively, wherein the number of fluorescent microspheres in the fifth droplet is 50, and the number of fluorescent microspheres in the fourth droplet and the sixth droplet is 0, then the droplet retardation value corresponding to the fifth droplet is the optimal droplet retardation value, and is 31.6. FIG. 1 shows the yields of fluorescent microspheres under the automatic droplet delay calculation and the manual droplet delay correction, wherein the abscissa is the droplet delay value, the ordinate is the sorting yield, the auto curve shows the yield under the automatic droplet delay calculation, and the manual curve shows the yield under the manual droplet delay correction, as shown in FIG. 1, the yield under the manual droplet delay correction condition is higher than the yield under the automatic droplet delay calculation.
Firstly, an instrument automatic droplet delay calculation method is used for calculating a droplet delay value, intervals of 0.1, 0.2 and 0.3 are respectively set manually, the droplet delay value is adjusted manually, about 50 fluorescent microspheres are collected under each droplet delay value (due to deviation of the droplet delay value, the number of the actually collected fluorescent microspheres is less than 50 fluorescent microspheres), and the obtained droplets are observed under a microscope, photographed and counted, and the yield is calculated. The results show that the drop delay has a very large influence on the yield, the drop delay values differ by 0.3, and the yield difference exceeds 20%, as shown in fig. 3; FIG. 3 is a statistical graph of the rate of yield loss for the case of deviation from the instrumentally automatically calculated droplet delay value of 0.3 for four experimental conditions; from this figure, it can be seen that under the four experimental conditions, when the droplet delay deviates from 0.3, the sorting yield decreases by 15% -30%, and the droplet delay affects the sorting yield.
Sorting fluorescent microspheres: 100 μm nozzle, sheath fluid pressure 30psi, sample pressure and sheath fluid pressure differential 0.3psi, purity mode selection, write envelope selection 1. Adjusting the optical path of the instrument, calculating the liquid drop delay by QC detection according to the method, respectively receiving the target microspheres and the central liquid flow according to the method reported in the literature (Riddell, Gardner et al 2015, A systematic adaptive evaluation method concrete performance using center stream catch), detecting the proportion of the microspheres in the collection liquid by using a flow cytometry, and calculating the R-max value. The target microsphere is a microsphere with a particle size of 3 mu m and medium fluorescence intensity and high fluorescence intensity; the central stream refers to the waste stream from the sorting process.
FIG. 2 is a graph showing R-max values and purities of two populations of medium fluorescence intensity and high fluorescence intensity among CST fluorescent microspheres sorted by R-max method. The CST fluorescent microspheres are calibration microspheres of a BD flow cytometer, and consist of 3 groups of fluorescent microspheres with different sizes and fluorescence intensities. FIG. 2(A) is a flow analysis diagram of two populations of microspheres collected by sorting and a central liquid stream, where Sample represents CST microspheres; CSC represents the central liquid stream; mid is a microsphere with medium fluorescence intensity; bright represents microspheres with high fluorescence intensity; delay is the drop delay, where the drop delay values are 30.96, 31.26, respectively; PE and FITC refer to fluorescein names for flow cytometry sorting analysis, wherein the PE is red, the PE is excited by a 488nm or 561nm laser, the absorption wavelength is 575nm, the FITC is green, the PE is excited by the 488nm laser, and the absorption wavelength is 513 nm; dim refers to a negative cell population; rmax refers to yield, fig. 2(B) is a graph of purity and yield of sorting according to data statistics analyzed by a flow cytometer, wherein the abscissa is a droplet delay value, the ordinate is a percentage of yield and a percentage of purity, bright precision indicates the purity of the microspheres with high fluorescence intensity, the purity of the microspheres with medium fluorescence intensity in midoutput is almost coincident, and as can be seen from fig. 2, the deviation of droplet delay has a great influence on sorting yield, but has almost no influence on purity; sorting two groups of microspheres with medium fluorescence intensity and highest fluorescence intensity in CST fluorescent microspheres by an R-max method; the yield is 97 percent and 95.7 percent respectively, the R-max value is 92.5 percent, and the R-max value obtained by the invention is superior to the value reported in the literature, which indicates that the separation method of the invention obtains higher separation yield.
Single cell sorting: single cell sorting was performed under the optimal droplet delay conditions described above. And setting sorting conditions: 100 μm nozzle, sheath fluid pressure 30psi, sample pressure and sheath fluid pressure differential 0.3psi, purity mode selected single, drop envelope selected 0.5. Adjusting the optical path of the instrument, passing quality control detection (QC detection), calculating the liquid drop delay according to the method, adjusting the sorter to an SD electroless sorting mode for sorting single cells of a 96-well plate and a 1536-well plate, pre-paving sheath liquid on the well plate by using the sorter, correcting the sorting position, then taking the single cell suspension of the double-stained jurkat cells in the step (2) for sorting, and sorting 1 cell in each well of the 1536-well plate.
(4) And (3) single cell yield identification: after sorting single cells into 1536 well plates, each well of the 1536 well plates was scanned with blue and green light from a BioTek rotation 1 multi-well plate cell imager, respectively, and recorded imagewise. And (3) confirming the cell condition in each hole by using double fluorescence, wherein a single cell is considered to be successfully sorted out when only one cell is doubly stained, the software is recorded as 1, other holes are recorded as 0, and the single cell yield is analyzed through data statistics. The yield of 1536 well plates was 53%. The culture is continued after the 96-well plate is used for sorting the single cells, and the sorting yield is calculated by counting the number of the formed single clones after one week. The yield (calculated as the number of monoclonal formations) of the 96-well plate was 91.6%.
(5) And (3) single cell activity identification: after the single cells are sorted to a 96-well plate, the single cells are placed in a carbon dioxide incubator to be continuously cultured, and the formation condition of the single cells is observed under a microscope about one week. Single cell viability was analyzed based on the single cell colony formation. The sorting receiving solution of the cell activity detection experiment is RPMI-1640 culture medium added with 10% FBS by volume fraction. The single cells obtained by sorting have high activity, and the monoclonal formation rate of a 96-pore plate reaches up to 91.6 percent.
Detection of sorted cells
(1) Detecting the apoptosis rate of the sorted cells: centrifuging the sorted cells received by different receiving solutions at a centrifugation speed of 300g for 3 minutes, discarding the supernatant, 500 mul of staining buffer (binding buffer, staining buffer carried in the existing apoptosis detection kit, the adopted kit comprises an Annexin V-FITC/PI apoptosis detection kit with the product number C1062M, the kit is resuspended with Annexin V dye, binding buffer and PI dye), centrifuging at a centrifugation speed of 300g for 3 minutes, 100 mul of staining buffer is resuspended, 5 mul of Annexin V dye is added and is shaded and stained for 15 minutes, 400 mul of staining buffer is added, 1 mul of diluted PI dye is added before the machine detection (wherein the PI dye is diluted by PBS buffer, the volume ratio of the PI dye to the PBS buffer is 1: 100, and the pH of the PBS buffer is 7.2-7.4), and the cell apoptosis condition is detected by using a double fluorescence channel.
FIG. 4 shows the results of apoptosis experiments with different receiving solutions. Specifically, 10 thousands of sorted cells are received by using an RPMI-1640 culture medium, an RPMI-1640 culture medium plus Fetal Bovine Serum (FBS) with a volume fraction of 10%, an RPMI-1640 culture medium plus FBS with a volume fraction of 50% and FBS with a volume fraction of 100%, the result graph of the staining of the apoptosis reagent and the statistic result of the apoptosis rate are shown in fig. 4, and fig. 4(A) -4 (D) are flow analysis detection graphs of the cells after being collected and sorted by four different receiving liquids and stained by an apoptosis detection kit respectively; FIG. 4(E) is a statistical plot of the apoptosis rate of cells sorted by four different receiving fluid collections. It can be seen from fig. 4 that the addition of FBS significantly reduced the apoptosis rate of the sorted cells, but the volume fractions of 10% FBS, 50% FBS and 100% FBS did not differ significantly.
(2) Proliferation assay of sorted cells:
and respectively taking the cells received by different sorted receiving solutions, counting, inoculating 2000 cells per well into a 96-well plate, adding 10 mu l of CCK8 reagent, respectively reading OD values of 450nm at 0 hour, 24 hours and 48 hours during plate laying, drawing a proliferation curve, and counting the cell proliferation condition.
FIG. 5 is a graph showing the results of a proliferation assay of cells after receiving sorting in four different receiving solutions; specifically, the sorted cells were received with RPMI-1640 medium (denoted by a), RPMI-1640 medium plus 10% FBS (fetal bovine serum) in volume fraction (denoted by b), RPMI-1640 medium plus 50% FBS in volume fraction (denoted by c), and 100% FBS in volume fraction (denoted by d), respectively; counting, adding CCK8 reagent after inoculation, reading OD values of 450nm at 24 hours and 48 hours respectively, and counting the cell proliferation, and as can be seen from FIG. 5, the proliferation capacity of the sorted cells can be improved after FBS is added into the receiving solution.
FIG. 6 is the statistical result of the yield and purity of 1536-well plate high-throughput single cell sorting under optimized conditions. In FIG. 6, the ordinate is the percent yield and percent Purity, Purity1, Purity2, Purity 3 are the Purity of three experiments, respectively, and the three curves are combined; the abscissa indicates: how much deviation, e.g. 0.0, compared to the optimal drop retardation value means 0, 0.2 means 0.2 deviation compared to the optimal drop retardation value, Yield of three experiments, Yield of Yield1, Yield2 and Yield 3. As can be seen from fig. 6, when high throughput sorting was performed, a phenomenon in which droplet delay affects the sorting yield of the well plate was also shown, and the yield of 1536 well plates was 53%. FIG. 7 is a monoclonal image of 96-well plate single cell sorting under optimized conditions; after optimizing the conditions, 88 wells of single cells were obtained per 96 well plate and monoclonals were formed within 7 days, as shown in fig. 7; the optimization condition specifically means that the optimal drop delay value is found by adopting the method, an experiment is carried out under the optimal drop delay value, the selected receiving solution is RPMI-1640 culture medium with 10% FBS by volume, and the drop falling position is adjusted to fall in the center of each hole of the hole plate.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (6)
1. A single cell sorting method with high yield and high activity is characterized by comprising the following steps:
(1) jurkat cell culture: jurkat cells were cultured in RPMI-1640 medium containing 10% FBS by volume and 1% p/s by volume, at 37 ℃ and 5% CO by volume2Culturing in an incubator, changing liquid every other day, carrying out passage for 3 days, discarding 4/5-volume cell suspension, adding an equal volume of fresh culture medium, and carrying out cell passage by the method;
(2) jurkat cell staining: taking the jurkat cell suspension obtained in the step (1), centrifuging 300g to remove a supernatant, adding 0.5ml of fresh culture medium for resuspension, dyeing by using a fluorescent dye CFSE (color filtration dye), dyeing for 20 minutes at room temperature in a dark place, and then dyeing for 5 minutes at 37 ℃ in a Horchest33342 in a dark place; centrifuging to remove supernatant, resuspending in PBS buffer solution and filtering with a 40 μm cell sieve to obtain double-stained jurkat cells for sorting;
(3) setting single cell sorting conditions: calibration of drop delay: firstly, finding a liquid drop delay value by using an instrument automatic liquid drop delay calculation method, and then further correcting the liquid drop delay value by using a manual liquid drop delay regulation method until the liquid drop delay value completely meets the condition of the optimal liquid drop delay, namely, continuously taking 10 liquid drops under different liquid drop delay values, wherein the number of target particles in the fifth liquid drop is the most, only the fifth liquid drop has the target particles, determining the liquid drop delay value at the moment as the optimal liquid drop delay value, and carrying out single cell sorting under the condition; and setting sorting conditions: a nozzle with the diameter of 100 mu m, the pressure of sheath fluid is 30psi, the pressure difference between sample pressure and sheath fluid is 0.3psi, the purity mode is selected to be single, and the drop envelope is selected to be 0.5; adjusting the optical path of the instrument, detecting QC, calculating the liquid drop delay according to the method, adjusting the sorter to an SD electroless sorting mode for sorting single cells of a 96-well plate and a 1536-well plate, pre-paving sheath liquid on the well plate by using the sorter, correcting the sorting position, then taking the cell suspension of the double-stained jurkat cells in the step (2) for sorting, and sorting 1 cell in each well of the 1536-well plate.
2. The method for sorting single cells with high yield and high activity according to claim 1, further comprising the step of single cell yield identification: after the single cells are sorted to a 1536-well plate, each well of the 1536-well plate is scanned by blue light and green light of a multi-well plate cell imager respectively, imaging records are carried out, the cell condition in each well is confirmed by double fluorescence, when only one double-stained cell exists, one single cell is considered to be successfully sorted, the score is 1, the score is 0 for other wells, and the yield of the single cells is counted.
3. The method of claim 1, further comprising single cell viability identification: sorting the single cells to a 96-well plate, placing the plate in a carbon dioxide incubator for continuous culture, and observing the formation condition of the single cells; single cell viability was analyzed based on the single cell colony formation.
4. The method of claim 2, wherein a BioTek rotation 1 multi-well plate cell imager is used for single cell yield determination.
5. A method for sorting single cells according to claim 3 with high yield and high activity, wherein in the single cell viability assay, sorted cells are received by receiving solution comprising RPMI-1640 medium plus 10% FBS by volume fraction.
6. Use of high yield high activity single cells in single cell sequencing, wherein said high yield high activity single cells are obtained by the sorting method according to any one of claims 1-5.
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