Mouse CD4⁺ T Cells Negative Selection Kit

Recommended Buffer (Not Provided)

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

Taking the isolation of CD4⁺ T cells from mouse spleen as an example

1. To prepare a single-cell suspension from a mouse spleen: Disaggregate the spleen using a 70 μm cell sieve, and rinse the sieve with pre-cooled PBS. Transfer the resultant cell suspension to a 50 mL centrifuge tube, centrifuge at 500 × g for 5 minutes, and discard the supernatant.

2. Add 5 mL of ACK Lysis Buffer to the tube, lysed at room temperature for 5 min. Add 20 mL of PBS to resuspend the cells, centrifuged at 500 × g for 5 min, and discard the supernatant.

Note: a. Erythrocyte lysis can be adjusted according to different lysates;
b. A small number of residual red blood cells will not affect subsequent sorting and purity.

3. Resuspend the cells in PBS; filter the cell suspension through a 70 μm cell sieve and count. Centrifuge the cells at 500 × g for 5 min and discard the supernatant.

Note: This step removes tissue or cell aggregates to avoid affecting the purity of separation.

4. Resuspend the cells in the Isolation Buffer, and adjust the cell density to 1 × 10⁸ cells/mL.

5. Transfer 100 μL of the cell suspension (1 × 10⁷ cells) to the bottom of a sterile tube. Add 2 μL of Biotin-Antibody Mix, mix well and incubate at 4°C for 10 minutes.

Note: a. Transfer the cell suspension directly to the bottom of the tube, rather than along the tube wall;
b. For larger cell quantities, proportionally increase the amount of Biotin-Antibody Mix.

6. To prepare the Streptavidin Magnetic Beads: Resuspend the Streptavidin Magnetic Beads thoroughly and transfer 20 μL of these beads to a 1.5 mL EP tube. Add 1 mL of Isolation Buffer, mix and centrifuge at 10,000 × g for 1 min. Discard the supernatant and repeat wash the beads 1-2 times, resuspend the beads in 20 μL of Isolation Buffer.

Note: The volume of Isolation Buffer used for the final resuspension should be equal to the initial volume of beads aspirated.

7. After cell-antibody incubation, add 20 μL of pre-washed beads to the tube, mix well and incubate at 4°C for 10 min.

Note: Proportionally increase the reagent amounts for larger cell quantities. For 5 × 10⁷ cells, add 10 μL of Biotin-Antibody Mix and 100 μL of beads to 500 μL of cell suspension. If the number of cells is less than 1 × 10⁷, adjust the cell suspension volume to 100 μL and add 2 μL of Biotin-Antibody Mix and 20 μL of beads.

8. After incubation, add 2.5 mL of Isolation Buffer to the tube and mix gently (avoid vigorous shaking or up-and-down mixing).

9. Place the tube in the magnetic separator for 5 min.

10. Carefully transfer the cell suspension into a sterile centrifuge tube (taking care to not detach the tube from the magnetic separator during this process). This tube contains the purified mouse CD4⁺ T cells. Centrifuge at 300 × g for 5 minutes, discard the supernatant and collect the cells.

Note: Avoid contact between the pipette tip and the magnetic beads; the cell suspension can also be collected by decantation.

11. According to the requirements of experiment wash the cells, and resuspend the cells in the appropriate buffer or medium. The cells can be used for downstream molecular or cell biology experiments.