Mouse Peripheral Blood Th17 Flow Cytometry Detection Experimental Procedure

1. Stimulation and Blocking (Stimulation and Blocking Kit E-CK-A091 required, self-provided)

1) Reagent Preparation (Before preparation, centrifuge the powder at 8000-10000 × g for 1 min to gather the powder at the bottom of the tube before dissolving and preparing):

a) 500× Cell Stimulation MIX: Add 100 μL of Cell Stimulation MIX Solvent to each tube of Cell Stimulation MIX Powder (50 μg), pipette and mix thoroughly to prepare 500× Cell Stimulation MIX.

b) 1000× Protein Transport Inhibitor MIX: Add 50 μL of 33% DMSO aqueous solution (self-provided) to each tube of Protein Transport Inhibitor MIX Powder (200 μg), pipette and mix thoroughly to prepare 1000× Protein Transport Inhibitor MIX.

2) Take 250 μL of fresh heparin-anticoagulated blood, add 250 μL of serum-free RPMI-1640 basal medium, and mix well.

3) Referring to the table below, add 500× Cell Stimulation MIX and 1000× Protein Transport Inhibitor MIX, and incubate the cells in a 37°C, 5% CO₂ incubator for 5-6 h, mixing once every 1-2 h during the incubation.

4) After incubation, collect the cells, centrifuge at 300 × g for 5 min, discard the supernatant, add 1 mL of Cell Staining Buffer [E-CK-A107] or 1× PBS (self-provided), mix well, centrifuge at 300 × g for 5 min, and discard the supernatant.

2. Red Blood Cell Lysis

5) Add 100 μL of Cell Staining Buffer [E-CK-A107] or 1× PBS to resuspend the cells, then add 2 mL of 1× ACK Lysis Buffer [E-CK-A105], and lyse on ice for 2-3 min. Centrifuge at 300 × g for 5 min, and discard the supernatant.

Note: Lysis is complete when the solution changes from turbid to clear. Centrifuge promptly to avoid excessive lysis that may cause cell damage.

6) Add 1 mL of Cell Staining Buffer [E-CK-A107] or 1× PBS to resuspend the cells, centrifuge at 300 × g for 5 min, and discard the supernatant.

7) Resuspend the cells in an appropriate volume of 1× PBS and perform cell counting, adjusting the cell density to approximately 1 × 10⁶ cells/mL.

3. Setting Up Sample Tube Grouping

4. (Optional) Amine-Based Viability Dye

8) According to the sample tube grouping, add 1 mL of cell suspension to each tube.

9) Add 1 μL of STYX™ Fixable Viability Dye (Note: Select an appropriate amine-based viability dye based on the flow cytometry panel) working solution per 1 mL of cell suspension, pipette gently to mix, and incubate at room temperature or 4°C in the dark for 30 min.

10) Centrifuge at 300 × g for 5 min, discard the supernatant, add 1 mL of PBS to resuspend the cell pellet, centrifuge at 300 × g for 5 min, and discard the supernatant.

11) Resuspend the cells in 100 μL of Cell Staining Buffer [E-CK-A107] (at this point, the cell density is approximately 1 × 10⁷ cells/mL).

5. Fc Receptor Blocking

12) Add 0.5-1 μg of purified anti-mouse CD16/32 monoclonal antibody [E-AB-F0997A] and incubate at room temperature for 10 min.

Note: No washing is required after incubation; surface antibodies can be added directly.

6. Surface Antibody Incubation

13) According to the experimental design, except for the blank control, add 5 μL of each corresponding surface staining antibody (CD3, CD4) to the single-stain tubes and the full-stain tube, mix well, and incubate at 4°C in the dark for 30 min.

7. Fixation and Permeabilization (Intracellular Factor Fixation and Permeabilization Kit E-CK-A109 required, self-provided)

14) Reagent Preparation: Permeabilization Buffer (5×) is a 5× concentrate. Before the experiment, dilute it with deionized water to prepare 1× Permeabilization Working Solution, i.e., take 1 mL of Permeabilization Buffer (5×) and add 4 mL of deionized water, mix well.

15) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube, centrifuge at 300 × g for 5 min, and discard the supernatant.

16) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.

17) Add 200 μL of Fixation Buffer to each tube, mix gently, and incubate at room temperature in the dark for 30-60 min.

Note: If the entire detection procedure cannot be completed on the same day, after fixation, wash the cells twice with PBS, resuspend in PBS or basal medium, and store at 4°C. Proceed with the remaining steps the next day.

18) Add 1 mL of 1× Permeabilization Working Solution, centrifuge at 600 × g for 5 min, and discard the supernatant.

8. Intracellular Antibody Incubation

19) Add 100 μL of 1× Permeabilization Working Solution to resuspend the cells.

20) Add 5 μL of intracellular antibody (IL-17A) to the corresponding single-stain tubes and the full-stain tube, and mix well.

21) Incubate at room temperature in the dark for 30 min.

22) Add 2 mL of Cell Staining Buffer [E-CK-A107] to each tube.

23) Centrifuge at 600 × g for 5 min, and discard the supernatant.

9. Flow Cytometry Detection

24) Add 200 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.

25) Adjust the instrument parameters and proceed with flow cytometry detection. Note: The voltage and threshold of the flow cytometer should be set before formal acquisition. Once signal acquisition has started, the voltage and threshold must not be adjusted.