2.3 Intracellular Factor Staining Case

1. Flow Cytometric Detection Protocol for Th1/Th2/Th17 in Human Peripheral Blood—PBMC Isolation Method
Experimental Equipment:

Flow cytometry tubes, 6-well cell culture plates, 37°C incubator, 5% CO₂ incubator, vortex mixer, palm centrifuge, horizontal rotor centrifuge, flow cytometer
Experimental Reagents:
Heparin sodium-anticoagulated whole blood, RPMI-1640 medium, FBS, lymphocyte separation medium, regular PBS,
PMA/Ionomycin + BFA/Monensin

2. Experimental Procedures
PBMC Isolation
Take 4 ml of fresh anticoagulated whole blood and isolate PBMCs using human peripheral blood lymphocyte separation medium.
Aspirate the PBMC layer. The PBMC layer is located below the plasma. Aspirate carefully and transfer to a 15 ml centrifuge tube.
Wash once with RPMI-1640 medium. Add RPMI-1640 to a total volume of 8 ml, invert to mix, and centrifuge at 20°C, 250×g for 10 minutes.
Discard the supernatant, resuspend the cells in 3 ml of RPMI-1640 medium (containing 10% FBS), and count the cells. Set aside for later use.
PBMC Stimulation
Take out the 6-well plate, label appropriately (unstimulated wells, stimulated wells), add 1 ml of PBMCs to each well, then add 1 ml of RPMI-1640 medium (10% FBS) to each well respectively, mix well, and avoid generating air bubbles.
Add 4 μl of stimulant to the stimulated wells, pipette to mix thoroughly, and minimize air bubbles.
Incubate in a 37°C, 5% CO₂ incubator for 5 hours.
After 5 hours, harvest the cells, centrifuge at 350×g for 5 minutes, and discard the supernatant.
After 5 hours of stimulation, harvest the cells; meanwhile, rinse the cell culture plate with 1 ml of RPMI-1640 medium, collect the rinse solution together into the centrifuge tube, centrifuge at 350×g for 5 minutes, and discard the supernatant.
Resuspend the cells in 2 ml of Staining Buffer each, centrifuge at 350×g for 5 minutes.
Discard the supernatant, resuspend the cells in 1 ml of Staining Buffer per tube respectively.
Blocking and Surface Staining
Aliquot the cells into tubes according to the unstimulated and stimulated groups, with 100 μl of cell suspension per tube.
Add 2.5 μg of Human Fc Receptor Blocker to each tube, mix well, and incubate at room temperature for 10 minutes.
Add the corresponding surface marker antibodies to each tube respectively, mix well, and incubate at room temperature in the dark for 15 minutes.
Add 2 ml of Staining Buffer to each tube to resuspend the cells, centrifuge at 350×g for 5 minutes.
Fixation, Permeabilization, and Intracellular Staining
Resuspend the fixed and permeabilized cells in 100 μl of Perm/Wash Buffer, add IL-4 and IFN-γ antibodies corresponding to each tube setup, and incubate at room temperature in the dark for 20 minutes.
Wash the cells twice with Staining Buffer. Centrifuge at 350×g for 5 minutes and discard the supernatant.
Resuspend the fixed and stained cells in 0.3 ml of Staining Buffer, and analyze by flow cytometry.
Case Flow Cytograms of Intracellular Flow Cytometric Detection of Th1/Th2/Th17 in Human Peripheral Blood:
Flow cytometric analysis of peripheral blood mononuclear cells (PBMC) using the Human Th1/Th2/Th17 Phenotyping Kit.
The staining patterns of IFN-γ, IL-17A, and IL-4 on resting PBMC (left column), PMA/Ionomycin-stimulated PBMC (middle column), and polarized Th17 cells (right column) are shown. The top three panels show the staining of IL-17A vs. IFN-γ, and the bottom three panels show the staining of IL-17A vs. IL-4. Dot plot analyses are derived from gated CD4⁺ cell populations. Flow cytometry was performed on an LSR II system.