Staining Protocol for Human Peripheral Blood Th1/Th2/Th17 Cells – PBMC Isolation Method

1. Required Equipment

Experimental instruments needed for the assay:

• Flow cytometry tubes
• 6-well cell culture plates
• 37 °C incubator with 5% CO₂
• Vortex mixer
• Mini centrifuge
• Horizontal rotor centrifuge
• Flow cytometer

2. Required Reagents

• Heparin sodium anticoagulated whole blood
• RPMI-1640 medium
• Fetal Bovine Serum (FBS)
• Lymphocyte separation medium
• Standard PBS
• PMA / Ionomycin, BFA / Monensin

3. Experimental Procedures

3.1 PBMC Isolation

1. Isolate PBMCs from 4 mL fresh anticoagulated whole blood using human lymphocyte separation medium, following the manufacturer’s official protocol.
2. Carefully aspirate the PBMC layer located beneath the plasma and transfer into a 15 mL centrifuge tube.
3. Wash cells once with RPMI-1640: top up to 8 mL with medium, invert to mix, centrifuge at 250 g for 10 min at 20 °C.
4. Discard supernatant, resuspend cell pellet in 3 mL RPMI-1640 supplemented with 10% FBS, perform cell counting for subsequent use.

3.2 PBMC Stimulation Culture

1. Label a 6-well plate into unstimulated control group and stimulated group. Add 1 mL PBMC suspension to each well, then supplement with 1 mL RPMI-1640 (10% FBS), mix gently without generating bubbles.
2. Add 4 μL stimulator (Cat. No. 550583) to each stimulated well, pipette gently to homogenize and avoid bubbles.
3. Culture the plate at 37 °C with 5% CO₂ for 5 hours.
4. After 5 h stimulation, collect cell suspension and centrifuge at 350 g for 5 min, discard supernatant.
5. Rinse each culture well with 1 mL RPMI-1640, combine rinse liquid with corresponding cell pellets, centrifuge at 350 g for 5 min and remove supernatant.
6. Resuspend pellets in 2 mL stain buffer, centrifuge at 350 g for 5 min.
7. Discard supernatant and resuspend each cell pellet in 1 mL stain buffer per tube.

3.3 Fc Blocking & Surface Marker Staining

1. Aliquot 100 μL cell suspension into separate tubes for unstimulated and stimulated groups respectively.
2. Add 2.5 μg human Fc blocking reagent to each tube, mix well and incubate at room temperature for 10 min.
3. Add matched surface marker antibodies, mix thoroughly, incubate at room temperature in dark for 15 min.
4. Dilute with 2 mL stain buffer and centrifuge at 350 g for 5 min to wash cells.

3.4 Fixation, Permeabilization & Intracellular Cytokine Staining

1. Resuspend fixed and permeabilized cells in 100 μL PermWash Buffer, add IL-4 and IFN-γ antibodies correspondingly, incubate at room temperature in dark for 20 min.
2. Wash cells twice with stain buffer, centrifuge at 350 g for 5 min each time, discard supernatant.
3. Resuspend final cell pellets in 0.3 mL stain buffer for flow cytometric acquisition and analysis.

4. Flow Cytometry Representative Plots

4.1 Human Peripheral Blood Th1/Th2/Th17 Intracellular Staining Plots

Figure 1. Flow cytometric analysis of human PBMCs stained with Human Th1/Th2/Th17 Phenotyping Kit. Dot plots gated on CD4⁺ T cells display IFN-γ, IL-17A and IL-4 expression in resting PBMCs (left), PMA/Ionomycin-stimulated PBMCs (middle), and polarized Th17 cells (right). Detected on BD™ LSR II flow cytometer.

4.2 Mouse Splenocyte Th1/Th2/Th17 Intracellular Staining Plots

Figure 2. Flow cytometric analysis of mouse splenocytes with Mouse Th1/Th2/Th17 Phenotyping Kit. Dot plots gated on CD4⁺ T cells show cytokine profiles of resting splenocytes (left), stimulated splenocytes (middle), and in vitro polarized Th17 cells (14-day polarization per Chen Dong et al.). Dead cell signals appear on diagonal; acquired on BD FACSCalibur™ System.