Mouse Spleen Treg Flow Cytometry Detection Experimental Procedure

1. Acquisition and Preparation of Spleen Samples

1) Sacrifice the mouse by cervical dislocation and place it on an operating table disinfected with 75% ethanol.

2) Spray an appropriate amount of 75% ethanol to moisten the mouse fur (to facilitate parting the fur and exposing the skin).

3) Place the mouse with its left side facing up. Use scissors to cut open the skin and peritoneum on the left side (at the mid-body position) to expose the internal organs. The spleen can be seen near the dorsal side as a dark red, elongated organ extending into the body cavity.

4) Use forceps to grasp one end of the spleen, slowly pull it outward, and use scissors to cut the adherent connective tissue until the spleen is completely detached.

5) Place the spleen tissue into a centrifuge tube containing PBS and store it on ice (to ensure the spleen tissue sample remains fresh).

6) Lay a layer of 200-mesh nylon filter mesh in a mortar and moisten it with a small amount of PBS.

7) Remove the spleen tissue, place it on the nylon mesh, and use a grinding pestle to repeatedly and gently press the spleen tissue until no obvious red tissue fragments remain (Note: Do not press too hard to avoid excessive cell rupture. A syringe plunger or other softer tools may be used instead of a glass grinding pestle to better protect the cells).

8) Rinse the nylon mesh with PBS and collect the dispersed cell suspension in the mortar (after rinsing, there should be no obvious red cells or particles remaining on the nylon mesh, only white adipose and other connective tissue residues).

9) Transfer the entire single-cell suspension from the mortar to a 15 mL centrifuge tube, rinse the mortar with PBS, combine the rinsing solutions, centrifuge at 300 × g for 5 min, and discard the supernatant.

10) Resuspend the cells in 2 mL of 1× ACK Lysis Buffer [E-CK-A105] and lyse at room temperature for 2 min.

Note: Lysis time should not exceed 2 min to prevent excessive lysis; lysis at 4°C is recommended.

11) After lysis is complete, immediately add 10 mL of PBS buffer (self-provided) to dilute the cell suspension and terminate the lysis.

12) Centrifuge at 300 × g for 5 min, discard the supernatant, then add an appropriate

13) volume of 1× PBS to resuspend the cells and perform cell counting. Adjust the cell density to 1 × 10⁶ cells/mL.

2. Setting Up Sample Tube Grouping

3. (Optional) Amine-Based Viability Dye

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

15) 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.

16) 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.

17) 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).

4. Fc Receptor Blocking

18) 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.

5. Surface Antibody Incubation

19) According to the experimental design, except for the blank control, add 5 μL of each corresponding surface staining antibody (CD4, CD25) to the single-stain tubes and the full-stain tube, mix well, and incubate at 4°C in the dark for 30 min.

6. Fixation and Permeabilization (Nuclear Factor Fixation and Permeabilization Kit E-CK-A108 required, self-provided)

20) Reagent Preparation: Fixation Concentrate (4×) is a 4× concentrate. Before the experiment, dilute it with Fixation Dilution Solution to prepare 1× Fixation Working Solution, i.e., take 1 mL of Fixation Concentrate (4×), add 3 mL of Fixation Dilution Solution, and mix thoroughly; the resulting mixture is 1× Fixation Working Solution.

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

22) Add 100 μL of Cell Staining Buffer [E-CK-A107] to resuspend the cells.

23) Add 1 mL of Fixation Working Solution to each tube, vortex to mix, and incubate at 4°C in the dark for 30 min. After the reaction, centrifuge at 600 × g for 5 min and discard the supernatant.

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 in the dark. Proceed with the remaining steps the next day.

24) Add 2 mL of Permeabilization Buffer to each tube, vortex to mix. Centrifuge at 600 × g for 5 min and discard the supernatant.

25) Repeat Step 23.

7. Nuclear Antibody Incubation

26) Add 100 μL of Permeabilization Buffer to resuspend the cells.

27) Add 5 μL of nuclear antibody (Foxp3) to the corresponding single-stain tubes and the full-stain tube, mix well, and incubate at room temperature in the dark for 30 min.

28) Add 2 mL of Permeabilization Buffer to resuspend the cells. Centrifuge at 600 × g for 5 min and discard the supernatant.

8. Flow Cytometry Detection

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

30) 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.