Mouse CD4⁺ CD25⁺ Regulatory T Cell Positive Selection Kit

Recommended Buffer (Not Provided)

Note: a. BSA may be replaced with human serum albumin (HSA) or 2% fetal bovine serum (FBS);
b. It is recommended to prepare the Isolation Buffer with water for injection, sterilize it through a 0.22 μm filter, and store it at 4°C.

Example: Isolation of mouse CD4⁺CD25⁺ regulatory T cells from mouse spleen or lymph nodes

I. Pre-enrichment of CD4⁺ T cells

1. Prepare a single-cell suspension from mouse spleen or lymph nodes using a 70 μm cell strainer and pre-cooled PBS. Centrifuge at 500 × g for 5 min and discard the supernatant.

2. Add 5 mL ACK Red Blood Cell Lysis Buffer and lyse at room temperature for 5 min. Add 20 mL PBS, resuspend the cells, centrifuge at 500 × g for 5 min, and discard the supernatant. Resuspend in PBS, filter through a 70 μm cell strainer, and count the cells.

3. Resuspend the cells in Isolation Buffer and adjust to 1 × 10⁸ cells/mL. Transfer 100 μL (1 × 10⁷ cells) and add 2 μL CD4 Enrichment Biotin-Antibody Mix. Mix thoroughly and incubate at 4°C for 10 min.

4. Thoroughly resuspend Streptavidin Magnetic Beads. Transfer 20 μL beads and wash 1-2 times with 1 mL Isolation Buffer (10,000 × g, 1 min). Finally resuspend in 20 μL Isolation Buffer.

5. Add 20 μL pre-washed Streptavidin Magnetic Beads to the antibody-incubated cells, mix thoroughly, and incubate at 4°C for 10 min.

6. Add 2.5 mL Isolation Buffer and mix gently. Place the tube on a magnetic separation rack for 5 min. While the tube remains on the rack, collect the supernatant, which contains pre-enriched CD4⁺ T cells.

7. Centrifuge the supernatant at 300 × g for 5 min, discard the supernatant, resuspend the cells in Isolation Buffer, and count the cells.

II. Positive selection of CD25⁺ cells

8. Adjust the pre-enriched CD4⁺ T cells to 1 × 10⁸ cells/mL. Transfer 100 μL (1 × 10⁷ cells), add 2 μL CD25 Capture Antibody, mix thoroughly, and incubate at 4°C for 10 min.

9. Transfer 20 μL Releasable Magnetic Beads, wash 1-2 times with 1 mL Isolation Buffer (10,000 × g, 1 min), and finally resuspend in 20 μL Isolation Buffer.

10. Add 20 μL pre-washed Releasable Magnetic Beads, mix thoroughly, and incubate at 4°C for 10 min.

11. Add 2.5 mL Isolation Buffer, mix gently, and place on a magnetic separation rack for 5 min. Discard the supernatant. Remove the tube, resuspend the bead-cell complexes in 2.5 mL Isolation Buffer, and perform magnetic separation again. Repeat for two washes.

12. Add 400 μL Magnetic Beads Release Buffer, gently resuspend, and incubate at room temperature for 10 min. Mix gently every 2-3 min during incubation.

13. Add 2.5 mL Isolation Buffer, mix gently, and place on the magnetic rack for 5 min. Collect the supernatant into a new sterile centrifuge tube; it contains bead-free CD4⁺CD25⁺ regulatory T cells.

14. Centrifuge at 300 × g for 5 min, collect the cells, wash as required, and resuspend in an appropriate buffer or culture medium for downstream experiments.