Mouse Spleen Th1/2 Cell Flow Cytometry Detection Experimental Procedure

1. Spleen Sample Preparation

1) Take a fresh mouse spleen sample and prepare it into a single-cell suspension. Centrifuge at 300 × g for 5 min and discard the supernatant.

2) Red Blood Cell Lysis: Add 2 mL of 1× ACK Lysis Buffer [E-CK-A105] to resuspend the cells, and let stand at room temperature for 2 min. Note: Lysis time should not exceed 2 min to prevent excessive lysis; lysis at 4°C is recommended.

3) After lysis is complete, immediately add 12 mL of PBS buffer (self-provided) or RPMI-1640 basal medium (self-provided) to terminate the lysis.

4) Centrifuge at 300 × g for 5 min, discard the supernatant, then add an appropriate volume of RPMI-1640 complete medium to resuspend the cells and perform cell counting. Adjust the cell density to 1 × 10⁶ cells/mL.

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

5) 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.

6) 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.

7) Collect the cell suspension, centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cell pellet.

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

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

3. Setting Up Sample Tube Grouping

4. (Optional) Amine-Based Viability Dye

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

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

12) 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.

13) 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

14) 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

15) 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)

16) 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.

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

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

19) 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.

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

8. Intracellular Antibody Incubation

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

22) Add 5 μL of each corresponding intracellular antibody (IFN-γ, IL-4) to the corresponding single-stain tubes and the full-stain tube, and mix well.

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

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

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

9. Flow Cytometry Detection

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

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.