Cytokineflow cytometry (CFC) is the collective term for flow cytometry that applies anti-cytokine antibodies to analyze cytokines that are markers of cell activation. The most common application of this technique is the labeling of intracellular cytokines in cells that have been activated, fixed and permeabilized for a short period of time in vitro. When stimulated with specific antigens, this technique allows for the quantitative analysis of rare antigen-specific T cells.
Principle
Cytokine flow cytometry is based on the principle of short-term in vitro activation with protein or peptide antigens or mitogens in the presence of a secretion inhibitor, treatment with EDTA to terminate activation and de-adherentize the cells, followed by fixation, staining with fluorescently-labeled antibodies, and analysis of the data with flow cytometry, with the results analysed by gating the cell population of interest (e.g., CD3+ CD8+ lymphocytes).
Appliance
Cytokine flow cytometry is commonly used to label intracellular cytokines in cells that have been activated and fixed and permeabilized for a short period of time in vitro.
Operation method
Flow cytometry analysis of cytokines
Principle
Cytokine flow cytometry is based on the principle of short-term in vitro activation with protein or peptide antigens or mitogens in the presence of a secretion inhibitor, treatment with EDTA to terminate activation and de-adherentize the cells, followed by fixation, staining with fluorescently-labeled antibodies, and analysis of the data with flow cytometry, with the results analysed by setting up gates to the cell populations of interest (e.g. CD3+CD8+ lymphocytes).
Materials and Instruments
Equipment: Move 1. Sample collection (1) For whole blood analysis: Collect whole blood with sodium heparin anticoagulation and store at room temperature for no longer than 8 h; (2) For fresh PBMC analysis: Separate PBMCs using the Ficoll gradient method or other commercially available methods (e.g., CPTTM tubes, BD Vacu-tainer, Franklin Lakes, NJ). resuspend PBMCs at a concentration of 2.5 × 106 to 1 × 107 viable lymphocytes/mL; (3) Analysis of frozen PBMCs: Thaw PBMCs quickly in a water bath at 37 ℃, slowly dilute with pre-warmed cRPMI-10 medium to 10 mL and centrifuge at 250 g for 7 min. Resuspend the cells in a small volume of pre-warmed cRPMI-10, count the viability of the cells by staining with Taipan blue, and dilute to a final concentration of 2.5 × 106 to 1 × 107 viable lymphocytes/mL; (4) Add 200 μL of whole blood or PBMCs per well in a 96-well plate. for whole blood or fresh PBMC analysis, proceed directly to Subheading 2. For frozen PBMC analysis, incubate at 37 ℃ overnight (12~18 h) before stimulation.
① 96-well tapered-bottom deep-well polystyrene plate and cover (BDDiscoveryLabware, Bedford, MA) (suitable for whole blood analysis)
② 96-well round-bottom standard polystyrene tissue culture plate and cover (suitable for PBMCs analysis)
③ 12-channel micropipette (35 mm for whole blood, 7 mm for PBMCs)
③ 12-channel micropipette (35 mm for whole blood, 7 mm for PBMCs)
⑤ Centrifuge capable of centrifuging deep-well plates (e.g. Sorvall Instruments, Newtown, CT)
⑥ Flow cytometer
⑤ Flow cytometer ⑦ Flow 96-well plate loading device
Reagents:
① Heparinized whole blood or PBMCs.
① Heparinized whole blood or PBMCs ② RPMI-1640 medium
① Heparinized whole blood or PBMCs ② RPMI-1640 medium ③ 20 mM HEPES
④ 10% fetal bovine serum
⑤ Antibiotic/antifungal solution (cRPMI-10)
⑥ Co-stimulatory antibodies (CD 28 and CD 49d)
⑦ In sterile phosphate buffer solution (PBS)
⑧ BrefeldinA stock solution
⑨ Dimethyl sulfoxide (DMSO)
⑩ EDTA
⑪ Erythrocyte lysate and cell fixation solution
⑫ Cell permeability reagent
⑬ Fluorescent labeled antibody
⑭ 0.5% bovine serum albumin
⑮ 0.1% sodium azide
⑯ Fixation solution
(1) Lysis of peptides or other antigens and Brefeldin A. Dilute peptide stock solution and 5 mg/mL brefeldin A stock solution 1:10 with PBS;
(2) Prepare a stimulant mix for each activated antigen. Generally the "Reagent Mix" should be slightly oversized (e.g., to stimulate 18 wells, prepare enough "Reagent Mix" for 20 wells);
(3) Pipette 20 μL of Reagent Mix into each well of cells and gently pipette to mix as described in Subheading 1;
(4) Cover the plate and incubate at 37 ℃ for 6 h. 3.
3. SampleWhole blood (deep-well plate)
(1) After activation, add 20 μL of 20 mM EDTA/PBS to each well and mix well by pipetting;
(2) Incubate at room temperature for 15 min and mix well by suction;
(3) Add 1.5 mL of room temperature 1 × BD FACS lysis solution to each well and incubate for 10 min at room temperature;
(4) Centrifuge at 500 g for 5 min, remove the supernatant with a 35 mm vacuum pipette, and resuspend the precipitate by aspiration with the remaining liquid;
(5) Add 1 mL of room temperature 1 × BD FACS permeabilization solution to each well and incubate for 10 min at room temperature;
(6) Add 0.5 mL of wash solution to each well, centrifuge the plate at 500 g for 5 min, and aspirate the supernatant with a 35 mm vacuum pipette;
(7) Add 1.5 mL of washing solution to each well, centrifuge the plate at 500 g for 5 min as above, and aspirate the supernatant;
(8) Resuspend the precipitate with appropriate titer of staining antibody mixture (e.g. anti-IFN-γ FITC/CD69 PE/CD8 PerCP-Cy5.5/CD3 APC). Incubate for 60 min at room temperature, avoiding light, and mix by blowing every 15~20 min;
(9) Add 1.5 mL of washing solution to each well, centrifuge the plate at 500 g for 5 min, and remove the supernatant with a 35 mm vacuum pipette;
(10) Resuspend the cells with 150 μL of 1% paraformaldehyde/PBS and store at 4 ℃ for 24 h under light protection until data acquisition.
PBMC (standard plate)
(1) Add 20 μL of 20 mM EDTA/PBS to each well and mix gently by pipetting;
(2) Incubate at room temperature for 15 min and mix well by pipetting;
(3) Centrifuge the plate at 250 g for 5 min and remove the supernatant with a 7 mm vacuum pipette;
(4) Resuspend the cells with 100 μL of 1 × BD FACS lysate per well and incubate for 10 min at room temperature;
(5) Add 100 μL of wash buffer to each well and centrifuge at 500 g for 5 min, and remove the supernatant with a 7 mm vacuum pipette;
(6) Resuspend the cell sediment with 200 μL of 1 × BD FACS permeabilization solution 2 per well and incubate for 10 min at room temperature;
(7) Centrifuge at 500 g for 5 min, and aspirate the supernatant with a 7 mm vacuum pipette;
(8) Add 200 μL of wash solution to each well, centrifuge the plate at 500 g for 5 min, and aspirate the supernatant with a 7 mm vacuum pipette;
(9) Add 200 μL of wash solution to each well, centrifuge the plate as in step 8, and discard the supernatant (a few more washes are necessary to remove residual permeabilizing fluid prior to antibody addition);
(10) Resuspend the cellular precipitate with an appropriate titer of antibody mix (e.g., anti-IFN-γFITC/CD69 PE/CD8 Per-CP-Cy5.5/CD3 APC). Incubate for 60 min at room temperature, avoiding light, and blow every 15-20 min;
(11) Add 200 μL of wash solution to each well, centrifuge the plate at 500 g for 5 min, and remove the supernatant with a 7 mm vacuum pipette.
(12) Add 200 μL of wash solution to each well, centrifuge the plate at 500 g for 5 min, and remove the supernatant with a 7 mm vacuum pipette;
(13) Resuspend the cell precipitate with 150 μL of 1% paraformaldehyde/PBS and store at 4 ℃ for 24 h under light protection until data acquisition.
4. Data Acquisition and Analysis(1) Calibrate the flow cytometer manually or with fluorescent microspheres and software;
(2) Manual calibration: set the value of each photomultiplier tube (PMT) with samples stained with isotype control mix, and set the compensation value with samples monolabeled with each fluorescein;
(3) Calibration with magnetic beads: set up according to the manufacturer's recommendations (e.g., Lyse No Wash and BD CalibriteTM microspheres with BD FACSComp software);
(4) Set the appropriate threshold for forward scatter to eliminate cellular debris and retain the lymphocyte population. If desired, set a second threshold in the CD3+ cell population to eliminate non-T cells and reduce the file size;
(5) Set collection criteria so that approximately 40,000 cells (e.g., CD4+ or CD8+ T cells) are collected per gate. When using an automated loading plate, set a secondary collection time standard so that the sample is not drained. For example, collect 200 μL of sample at high speed on a BD FACS CaliburTM and set the collection time limit to a maximum of 180 s;
(6) Store the collection template and machine parameters after several samples have been collected to calibrate the settings and set the gate;
(7) If feasible, enter all appropriate information into the autosampling software;
(8) Obtain data;
(9) Analyze the data by forward and side-scattering to define small lymphocyte zones and other corresponding cell populations (e.g., CD3+CD8+ cells) with appropriate reagents. Take care to appropriately size these zones to include cells whose activation results in downregulation of CD3 and CD8 (or CD4);
(10) Create a scatter plot by the two selected regions showing cytokine staining with CD69;
(11) Using a positive control sample (e.g., SEB-stimulated), delineate a "response zone" that includes all cells that are double positive for CD69 and cytokines. If an automated gate is used, the response zone is customarily delineated as a double-negative cluster (automatically defined by the gate);
(12) The percent positivity of each corresponding region for each sample is recorded, and the net antigen-specific response value is obtained by subtracting the corresponding number of non-stimulated cells (negative control) from the percent positive cells for each stimulated sample;
(13) If desired, the percentage can be converted to an absolute number of reactive cells per milliliter of blood by multiplying by the donor's count per milliliter of CD4 or CD8.
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