Human Peripheral Blood Th1/Th2/Th17 Subset Detection

1. Experimental Materials

1.1 Laboratory Equipment

• Consumables & Instruments: Flow cytometry tubes, 6-well cell culture plates
• Incubation System: 37°C, 5% CO₂ cell incubator
• Processing Devices: Vortex mixer, palm microcentrifuge, horizontal rotor centrifuge
• Detection Platform: Flow cytometer

1.2 Experimental Reagents

• Heparinized whole blood
• RPMI-1640 medium, fetal bovine serum (FBS)
• Human peripheral blood lymphocyte separation medium
• Sterile phosphate buffered saline (PBS)
• Stimulant cocktail: PMA/Ionomycin + BFA/Monensin

2. Standard Protocol

2.1 PBMC Isolation

1. Transfer 4 mL fresh heparinized whole blood and isolate peripheral blood mononuclear cells (PBMCs) using lymphocyte separation medium.
2. Carefully aspirate the PBMC layer located beneath the plasma and transfer the cell suspension into a 15 mL centrifuge tube.
3. Dilute cells to 8 mL total volume with RPMI-1640 medium, invert to mix, and centrifuge at 250×g, 20°C for 10 min.
4. Discard supernatant, resuspend cell pellet in 3 mL RPMI-1640 supplemented with 10% FBS, count viable cells and set aside for subsequent stimulation.

2.2 PBMC Stimulation

1. Label a 6-well plate as unstimulated control and stimulated groups. Add 1 mL PBMC suspension to each well, then supplement with 1 mL 10% FBS RPMI-1640; mix gently without bubble formation.
2. Add 4 μL stimulant cocktail to each stimulated well, pipette thoroughly to mix and minimize air bubbles.
3. Incubate the plate at 37°C under 5% CO₂ for 5 h.
4. After incubation, collect cells into centrifuge tubes and centrifuge at 350×g for 5 min; discard supernatant.
5. Rinse each well with 1 mL RPMI-1640, combine rinse solution with corresponding cell pellets, centrifuge at 350×g for 5 min and remove supernatant.
6. Resuspend pellets in 2 mL Staining Buffer, centrifuge at 350×g for 5 min, discard supernatant, and reconstitute each tube with 1 mL Staining Buffer.

2.3 Fc Blocking & Cell Surface Staining

1. Aliquot cell suspension into separate tubes for control and stimulated groups, 100 μL per tube.
2. Add 2.5 μg human Fc receptor blocking reagent to each tube, mix and incubate at room temperature (RT) for 10 min to block non-specific antibody binding.
3. Add matched surface marker antibodies to respective tubes, mix well, and incubate in the dark at RT for 15 min.
4. Dilute with 2 mL Staining Buffer, centrifuge at 350×g for 5 min, discard supernatant.

2.4 Fixation, Permeabilization & Intracellular Cytokine Staining

1. Resuspend fixed-permeabilized cell pellets in 100 μL Perm/Wash Buffer, add IFN-γ and IL-4 antibodies per tube layout, and incubate protected from light at RT for 20 min.
2. Perform two washes with Staining Buffer; each wash consists of 350×g centrifugation for 5 min followed by supernatant removal.
3. Final resuspension: reconstitute stained cells in 0.3 mL Staining Buffer for flow cytometric acquisition.

3. Data

All intracellular cytokine staining data were acquired using the Human Th1/Th2/Th17 Phenotyping Kit on an LSR II flow cytometer. All dot plots were gated on CD4⁺ T lymphocyte populations.

Three sample groups are displayed in columns from left to right: resting unstimulated PBMCs, PMA/Ionomycin-stimulated PBMCs, and fully polarized Th17 cells. Two plot panels are provided per group:

• Top row: IL-17A vs IFN-γ co-expression
• Bottom row: IL-17A vs IL-4 co-expression