A FLOW CYTOMETRIC DETECTION METHOD FOR LYMPHOCYTE IN IMMUNE CELLS

Abstract

The present application relates to a flow cytometric detection method for lymphocytes in immune cells. The method includes steps of: a) lymphocyte samples stained with immunofluorescent antibodies were added into the pre-cooled PBS-EDTA solution, and the cells were mixed and prepared for flow cytometry detection; b) starting up and warming up a flow cytometer system, adjusting the flow speed, then adding PBS-EDTA solution into a sample tube, and flushing a nozzle system of liquid stream; c) the sample of homogenous cells obtained in step a) is added to the sample tube and then tested. The present detection method can separate cell subpopulations, so that the analysis and detection are more accurate, and is especially suitable for detecting cell subpopulations with small number of cells, thereby saving antibodies and reagents.

Claims

1. A method for detecting lymphocytes in immune cells, comprising the steps of: a) lymphocyte samples stained with immunofluorescence antibody were added into the pre-cooled PBS-EDTA solution, and the cells were mixed and prepared for flow cytometry detection; b) starting up and warming up a flow cytometer system, adjusting the flow speed to a low-speed range of 10-29 μl/min or a medium speed range of 30-44 μl/min, then adding PBS-EDTA solution into a sample tube, and flushing a nozzle system of liquid stream; c) the sample of homogenous cells obtained in step a) was added to the sample tube and then tested.

2. The method according to claim 1, wherein the PBS-EDTA solution is a mixed solution containing 0.005-0.05M PBS and a final concentration of 2-3 mM EDTA with a pH of 7.2-7.4.

3. The method according to claim 1, wherein step a) further includes: the concentration of cells in the lymphocyte sample is 1×10.sup.6-10×10.sup.6/ml.

4. The method according to claim 1, characterized in that, step a) further includes: the volume of the lymphocyte sample is 50-200 μl.

5. The method according to claim 1, wherein in step b), the flow speed is adjusted to a medium speed range of 30-44 μl/min; preferably a medium speed range of 35-40 μl/min.

6. The method according to claim 1, wherein step b) further includes a preheating time of 5-10 minutes.

7. The method according to claim 1, wherein step c) further includes setting conditions of 3000-20000 cells in the gate, preferably 15000 cells in the gate, and collecting samples.

8. The method according to claim 1, wherein, the flow cytometric cell sample is prepared by the following method: 1) in vitro anticoagulant blood samples were taken for centrifugation to separate plasma and blood cell precipitation to obtain blood cell precipitation; 2) blood cell precipitation was diluted with PBS solution to obtain blood cell diluent; 3) the blood cell diluent was slowly added to a centrifuge tube containing an equal volume of lymphocyte separation solution, and then centrifuged to remove the plasma to obtain the middle buffy coat cells diluent; 4) adding the buffy coat cells obtained in step 3) into a centrifuge tube, adding PBS solution under stirring to obtain a suspension, centrifuging, removing supernatant, and then adding PBS solution to adjust the cell concentration to 1×10.sup.5/ml-1×10.sup.8/ml, preferably 1.5×10.sup.6/ml-5×10.sup.6/ml, further preferably 1.5×10.sup.6/ml-3.0×10.sup.6/ml; 5) adding the lymphocyte cells obtained in step 4) after adjusting the concentration thereof into a flow tube; and adding flow cytometric antibody to label lymphocyte subpopulations, evenly mixing, standing at 2-8° C. and incubating without light; 6) adding PBS solution, mixing, and centrifuging to remove unbound antibody; 7) completion of samples {circle around (1)} If a detection on a flow cytometer is performed immediately, the supernatant is discarded and the cells are evenly mixed with PBS-EDTA solution pre-cooled to 2-8° C. for flow cytometric detection; {circle around (2)} If the detection cannot be performed immediately, cells are directly resuspended by adding 0.05-5% formalin, mixed, and left to stand at 2-8° C. in the dark; and before detection, each of the tubes is added with PBS solution, centrifuged to discard the supernatant, and added with PBS-EDTA solution at 2-8° C. to evenly mix cells for flow cytometric detection.

9. The method according to claim 8, wherein the PBS solution is 0.005-0.05M PBS solution at pH 7.2-7.4; and the PBS-EDTA solution is a mixed solution at pH 7.2-7.4 containing 0.005-0.05M PBS and a final concentration of 2-3 mM EDTA.

10. The method according to claim 8, wherein step 1) further comprises centrifuging conditions of 1500-3500 rpm for 5-30 min, preferably 1500-2900 rpm for 15-20 min.

11. The method according to claim 8, wherein step 2) further comprises diluting blood cell precipitate with PBS solution by a volume ratio of 1:0.5 to 1:2, preferably 1:1.

12. The method according to claim 8, wherein step 3) further comprises centrifuging conditions of 1500-3500 rpm, 10-20 min and 4° C.; preferably, 1500-2900 rpm, 15-20 min and 4° C.

13. The method according to claim 8, wherein step 4) further comprises centrifuging conditions of 1500-3500 rpm, 5-20 min and 4° C.; preferably centrifuging conditions of 1500-2900 rpm, 5-10 min and 4° C.

14. The method according to claim 8, wherein PBS solution is added to the centrifuge tube in step 4) until the volume is 10 ml-15 ml.

15. The method according to claim 8, wherein step 6) further comprises centrifuging conditions of 1500-2900 rpm for 5-10 min.

16. The method according to claim 8, wherein the amount of PBS solution added in step 6) is 2 ml.

17. The method according to claim 8, wherein the PBS-EDTA solution in step 7) is an EDTA mixed solution at pH 7.2-7.4 containing 0.005-0.05M PBS and a final concentration of 2-3 mM EDTA; and the PBS solution is 0.005-0.05M PBS solution at pH 7.2-7.4.

18. The method according to claim 8, wherein the centrifuging conditions in step 7) are 1500-2900 rpm for 5-10 min.

19. The method according to claim 8, wherein the method comprises: (I) Isolation of Human Peripheral Blood Mononuclear Cells from Peripheral Blood (1) 0.2 mL of the isolated original blood sample was taken for counting, and 1 mL of the sample was retained with labeled information and stored at 4° C.; (2) centrifuging the blood in a centrifuge tube at 1500-2900 rpm for 15-20 minutes, and storing the upper plasma in a 2 ml cryopreservation tube at −80° C.; (3) diluting the cell precipitate of the rest of the blood with PBS solution by a volume ratio of 1:1; (4) prepare a 15 ml centrifuge tube and fill the pipette with lymphocyte separation solution equal to the blood cell diluent; (5) slowly add the blood cell diluent to the centrifuge tube in step (4), keeping the separation fluid well stratified; (6) placing the centrifuge tube into a low-speed centrifuge, balancing, and centrifuging at 1500-2900 rpm for 15-20 mins; (7) after centrifugation, the upper plasma was slowly absorbed with a 10 ml pipette or a 3 ml Pasteur pipette and discarded; (8) pipetting middle buffy coat cells by circles into a new 15 ml centrifuge tube, adding PBS solution to a volume of 10 ml, counting 200 μl of cell suspension on a whole blood cell counter for subsequent adjustment of cell concentration, and centrifuging the rest of the cell suspension at 1500-2900 rpm for 5-10 mins; and (9) centrifuging, discarding the supernatant, and, according to the result of counting, adding appropriate amount of pre-cooled PBS solution at 2-8° C. to adjust the cell density to 1.5-2.0×10.sup.6/ml; (II) Preparation of flow cytometric samples (10) sample staining Each of the flow tubes is added with 100 μl cell suspension, added with corresponding flow cytometric antibody so that the antibody is evenly mixed with cells, and incubated in the dark at 4° C. for 10-30 minutes, preferably 15-20 minutes and most preferably 20 minutes. (11) sample washing and solution adding After centrifuging, the supernatant is discarded, and the cells are resuspended in 2 ml PBS solution at 2-8° C., and centrifuged at 1500-2900 rpm for 5-10 min to discard supernatant after centrifuging; {circle around (1)} If a detection on a flow cytometer is performed immediately, the cells are evenly mixed with 400 μl pre-cooled PBS-EDTA solution ready for flow cytometric detection; {circle around (2)} If the detection cannot be performed immediately, cells are resuspended in 500 μl 0.05-5% formalin, vertex mixed, and stored at 4° C. in the dark; and before detection, each of the tubes is added with 2 ml PBS solution, centrifuged at 1500-2900 rpm for 5-10 min to discard the supernatant, and added with 400 μl pre-cooled PBS-EDTA solution to evenly mix cells for flow cytometric detection.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0072] FIG. 1: detection diagram of lymphocyte subsets in Example 2;

[0073] FIG. 2: detection diagram of lymphocyte subsets in Example 2;

[0074] FIG. 3: detection diagram of lymphocyte subsets in Example 3;

[0075] FIG. 4: detection diagram of different lymphocyte subsets in example 3;

[0076] FIG. 5: detection diagram of DC cell subtypes in Example 3;

[0077] FIG. 6: comparison of cell populations of the sample obtained by the method of Example 1 and the sample obtained by the lysis method in the literature in Example 4; (the upper three figures show the flow cytometric diagrams obtained by the lysis method, and the lower three figures show the flow cytometric diagrams obtained by separation of corresponding samples in the upper three figures.)

[0078] FIG. 7: comparison diagram of cell enrichment between the sample obtained by the method of Example 1 and the sample obtained by the lysis method in the literature in Example 4;

[0079] FIG. 8: comparison diagram of enrichment of different types of cells between the sample obtained by the method of Example 1 and the sample obtained by the lysis method of literature in Example 4.

DETAILED DESCRIPTION

[0080] The following embodiments and test examples are used to further elaborate the application, not intended to limit the effective scope of the present application in any way.

Example 1 Preparation of PBMC Samples Needed for Flow Cytometry

[0081] 1. Purpose

[0082] preparing PBMC samples needed for flow cytometric detection (also referred to as “samples” in the context of the present application)

[0083] 2. Principle

[0084] There are relatively complete antigens or receptors remained on the surface of living cells. Fluorescent labeled antibodies are used to bind to corresponding antigens on the surface of cells, and fluorescence intensity and positive percentage are measured to provide the density and distribution of corresponding antigens.

[0085] 3. Preparation of Instruments and Consumable Materials

[0086] 3.1 Instruments

[0087] Super Clear Workbench, Era Beili low speed centrifuge DT5-4, a vortex mixer, pipettes of various ranges, and a blood cell counter

[0088] 3.2 Consumable Materials

[0089] 15 ml centrifuge tube, 10 ml pipette, 1.5 ml centrifuge tube, a flow tube, 3 ml pasteurizer pipette, and pipette tips of various ranges.

[0090] 3.3 Formulations of Reagents

TABLE-US-00001 Name Stock solution Formulation 1× PBS 10× PBS stock solution: purified water = 1:9 0.1% formalin 10% formalin stock solution:1× PBS = 1:9 PBS solution 1× PBS 1× PBS (0.01M PBS, pH = 7.2-7.4) 1× hemolysin 10× hemolysin stock solution: purified water = 1:9 PBS-EDTA 1× PBS adding 0.5M EDTA to 1× solution PBS (0.01M PBS; pH = 7.2-7.4) until the final concentration of EDTA is 2.5 mM

[0091] Routine flow cytometric antibodies;

[0092] 4. Operation Procedure (Illustrated by Taking 5 ml In Vitro Original Blood Sample as an Example)

[0093] 4.1. Separation of In Vitro Peripheral Blood Sample (PBMC)

[0094] (1) obtaining 0.2 ml in vitro original blood sample and counting;

[0095] (2) adding 5 ml blood to a 15 ml centrifuge tube, centrifuging at 1500-2900 rpm for 15-20 min, and discarding upper plasma;

[0096] (3) diluting cell precipitate of the rest of the original blood with PBS solution by volume;

[0097] (4) providing a centrifuge tube and adding lymphocyte separation solution having a volume equal to the volume of the blood cell diluent to the centrifuge tube by using a pipette;

[0098] (5) adding blood cell diluent into the centrifuge tube in step (4) slowly for keeping the layer of the separation solution clear;

[0099] (6) slowly placing the centrifuge tube into a centrifuge, balancing, and centrifuging at 1500-2900 rpm for 15-20 min;

[0100] (7) slowly suctioning and discarding the supernatant with a pipette after centrifuging;

[0101] (8) pipetting middle buffy coat cells into a new centrifuge tube, adding PBS solution to a volume of 10 ml, counting 200 μl of the cell suspension on a blood cell counter, and centrifuging the rest of the cell suspension at 1500-2900 rpm for 5-10 min;

[0102] (9) discarding the supernatant after centrifuging, and removing residual liquid by using a pipette. According to the result of counting, appropriate amount of pre-cooled PBS solution at 4° C. was added, so as to adjust the density of cells to 1.5-3.0×10.sup.6/ml, for example, 2×10.sup.6/ml.

[0103] 4.2 Preparation of flow cytometric samples (the temperature was controlled in this operation, and the samples and antibodies were operated in an ice box throughout the operation)

[0104] (1) Sample Staining

[0105] A flow tube was added with 100 μl lymphocyte suspension, added with flow cytometric antibody, gently shook in a vortex mixer to fully mix the antibody with the cells, and incubated in the dark at 2-8° C. for 20 minutes.

[0106] (3) Sample Washing

[0107] After incubating, the supernatant was discarded, and the cells were resuspended in 2 ml pre-cooled PBS solution, and centrifuged at 1500-2900 rpm for 5-10 minutes.

[0108] (4) Completion of Samples

[0109] {circle around (1)} If the detection on a flow cytometer is performed immediately, the supernatant is discarded and the cells are mixed in 400 μl pre-cooled PBS-EDTA solution for flow cytometric detection;

[0110] {circle around (2)} If the detection cannot be performed immediately, cells are suspended in 500 μl 0.05-5% formalin, vortex mixed, and left to stand at 4° C. in the dark. Before detection, each of the tubes is added with 2 ml PBS solution, and centrifuged at 1500-2900 rpm for 5-10 minutes to discard the supernatant, and the cells are mixed with 400 μl pre-cooled PBS-EDTA solution for detection on a flow cytometer.

[0111] 5. Notes

[0112] 5.1 the antibodies and cells should be kept at low temperature (4° C.) during the operation.

[0113] 5.2 after adding the antibodies, the antibodies and the cells should be fully mixed.

Example 2 Detection of PBMC Samples

[0114] 2.1 Instruments, Reagents and Samples:

[0115] Sample: prepared by the method of Example 1 from in vitro blood samples;

[0116] Flow cytometer: ACEA NovoCyte

[0117] 2.2 Detection Method:

[0118] a) checking the status of the cytometer before starting up;

[0119] b) warming up the flow cytometer system for 5-10 minutes and performing perfusion (for about 15 minutes);

[0120] c) performing quality control procedure and proceeding to the next step after qualification;

[0121] d) setting the parameters of the flow cytometer, adjusting the voltage of each of detection channels, and setting the liquid flow speed to 35-40 μL/min;

[0122] e) vortex vibrating the prepared flow cytometric samples for 3 seconds, and subjecting the samples to detection under the conditions of: 15000 cells in the gate; and collecting samples.

[0123] 2.3 Results of Detection

[0124] It can be seen from FIG. 1 that, in the plot of FSC vs SSC, gate 1: lymphocyte population, lymphocyte subpopulations can be clearly identified, and the data will be more reliable and accurate since it is populations of cells that are main analyzed.

[0125] It can be seen from FIG. 2 that: (1) gate 1 is selected to display FL1 and FL3 area in gate 1; (2) select cross gate 2 in the gate 1: CD3+CD4+ cell population (Q2 region) is selected. The lymphocyte subpopulations to be analyzed are well separated, the gate is more easier to set, and the data is more accurate.

Example 3 Detection of PBMC Samples

[0126] 3.1 Instruments, Reagents and Samples:

[0127] Sample: prepared by the method of Example 1 from 5 ml in vitro blood samples; Flow cytometer: ACEA NovoCyte

[0128] 3.2 Detection Method:

[0129] a) checking the status of the cytometer before starting up;

[0130] b) warming up the flow cytometer system for 5-10 minutes and performing perfusion (for about 15 minutes);

[0131] c) performing quality control procedure and proceeding to the next step after qualification;

[0132] d) setting the parameters of the flow cytometer, adjusting the voltage of each of detection channels, and setting the liquid flow speed to 35-40 μL/min;

[0133] e) vortex vibrating the prepared flow cytometric samples, and subjecting the samples to detection under the conditions of: 15000 cells in the gate; and collecting samples.

[0134] 3.3 Results of Detection

[0135] 1) FIG. 3 shows the analysis of lymphocyte subpopulations obtained from the peripheral blood of one healthy person by lymphocyte separation method, in which a population of lymphocyte is in circled gate P1. As can be seen from FIG. 3, the background is clean, and the population of lymphocyte and population of cell debris are well and ideally separated.

[0136] It can be seen that, the present application separates and enriches lymphocytes by using lymphocyte separation solution, which are obviously divided, ensuring reliable and accurate detection results while reducing antibodies and reagents.

[0137] 2) FIG. 4 shows that, through the detection of enriched lymphocyte subpopulations, lymphocyte subpopulations with relatively small number of cells are effectively analyzed, and also the specific lymphocyte subpopulations can be effectively stained; In FIG. 4, for sample 4 on the left, Q3-2 is CD8+T cells, which are obviously separated via separation and enrichment so that the subpopulations can be well analyzed. The right figure of FIG. 4 shows the cell subpopulations with a small proportion of lymphocyte subpopulations, that is, DC cell subpopulation, which can also be accurately analyzed after enrichment.

[0138] 3) FIG. 5 clearly defines two subtypes of DC cells, which otherwise should not be identified in conventional lymphocyte subpopulation analysis. The two subtypes of cells were enriched by separation. As shown in the right 1 of FIG. 5, the population is obviously separated and the boundary is clear.

Example 4 Comparison Between the Sample Prepared in the Method of the Present Application and the Sample Obtained by the Hemolysin Lysis Method

[0139] 4.1 Samples and Reagents:

[0140] The lymphocyte sample of the present application: prepared by the method of Example 1 from in vitro peripheral blood sample;

[0141] Comparative lymphocyte samples: prepared by the method according to following section 4.2.1;

[0142] 5 ml anticoagulant blood sample;

[0143] 1×PBS: prepared from 10×PBS solution: pure water=1:9 by volume;

[0144] PBS solution: 1×PBS;

[0145] PBS-EDTA solution: prepared by adding 0.5m EDTA to 1×PBS until the final concentration of EDTA is 2.5 mM;

[0146] Conventional flow cytometric antibodies: anti-CD3 antibody, lin1[Lineage cocktail 1], anti-CD123 antibody, anti-CD11 antibody, CD3 FITC, CD8PerCP, CD4PerCP.

[0147] 4.2 Testing Method:

[0148] 4.2.1 Hemolysin Lysis Method:

[0149] (1) Sample staining: adding 100 μl anticoagulant blood sample into each of flow tubes, and adding flow cytometric antibody to label lymphocyte subpopulations respectively, so that the antibody was fully mixed with cells, and incubating the flow tubes in the dark at 2-8° C. for 20 minutes.

[0150] (2) Lysis: adding 2 ml 1×BD hemolysin (i.e. red blood cell lysis solution) to the flow tube, and performing lysis for 5 minutes.

[0151] (3) Sample washing: 5 minutes later, centrifuging at 1500-2900 rpm for 10 minutes, discarding the supernatant, adding 2 ml pre-cooled PBS solution to resuspend the cells, centrifuging at 1500-2000 rpm for 5-10 minutes, discarding the supernatant, adding 400 μL PBS-EDTA solution at 2-8° C., and evenly mixing.

[0152] 4.2.2 Method for Detection on a Flow Cytometer

[0153] a) before starting up, checking the volume of sheath barrel, waste liquid barrel, detergent and flushing agent, adding enough sheath liquid, and pouring out waste liquid;

[0154] b) warmed up the flow cytometric system for 5-10 minutes and perform perfusion (for about 15 minutes);

[0155] c) performing quality control procedure and proceeding to the next step after qualification;

[0156] d) setting the parameters of the flow cytometer, adjusting the voltage of each of detection channels, and setting the liquid flow speed to 35-40 μL/min;

[0157] e) vortex vibrating the prepared flow cytometric samples of the present application and comparative lymphocyte samples for 3 seconds, respectively, and subjecting the samples to detection under the conditions of: 15000 cells in the gate; and collecting samples.

[0158] 4.3 Results of Experiments:

[0159] 4.3.1 through comparison, it can be seen that there is much debris in the sample obtained by lysis, and the background is not clean, which influences the collection and analysis of flow cytometry data, and the compensation adjustment of lymphocytes may cause bias phenomenon; and the present method (method by using lymphocyte separation solution) provides clean background and clear and obvious populations (see FIG. 6).

[0160] In particular, for the lymphocytes in the gate circled in the lymphocyte subpopulation analysis obtained by lysis method, it can be seen from the figure that the lymphocyte subpopulation and the cell debris population is not ideally separated (see the three figures in the first row of FIG. 6).

[0161] For the lymphocytes in the gate circled in the lymphocyte subpopulation analysis obtained by the present method, it can be seen for the figure that the lymphocyte subpopulations and cell debris population are well and ideally separated (see the third figures in the second row of FIG. 6).

[0162] 4.3.2 It can be seen from FIG. 7 that, in the present method, when the fluorescent antibody is used, since the number of lymphocytes is counted and the fluorescent antibody is added in proportion, more than 70% of the antibody is saved. The amount of combined antibody used in the present application is 15 μL, which is 60 μL in the lysis method. The method of the present application does not cause excessive or insufficient antibody. It can be seen from FIG. 7 that CD3 FITC and CD8PerCP were labeled in group 1, and CD3 FITC and CD4PerCP were labeled in group 2. Due to the different samples obtained by different treatment methods, for the same separated peripheral blood sample, the result showed that, the proportion of double positive groups in the sample obtained by the lysis method is significantly reduced, and the amount of antibody cannot be quantified; while for the sample obtained by the method of the present application, the antibody can be quantitatively added according to the amount of cells. In addition, it is obvious that the cells obtained by the separation method are significantly enriched.

[0163] 4.3.3 it can be seen from FIG. 8 that the sample prepared by the present application has enrichment effect for the sample with small proportion of cell subpopulations. For the same separated peripheral blood sample, for the MDC and PDC accounting for small proportion, the observation results of the samples as obtained are different in the lysis method and the method of the present application, and the cell subpopulations in the samples obtained by the present application are enriched, which can be shown from the number and percentage of cells.