Mouse CD3 Cell Depletion Magnetic Beads (Positive Selection)

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: Depletion of CD3⁺ cells from a mouse spleen or other tissue single-cell suspension

1. Prepare a mouse spleen or other tissue single-cell suspension and perform red blood cell lysis as appropriate for the sample type. Filter through a 70 μm cell strainer and count the cells. Centrifuge at 500 × g for 5 min and discard the supernatant.

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

3. Thoroughly resuspend the CD3 Cell Depletion Magnetic Beads. Transfer 20 μL beads to a 1.5 mL microcentrifuge tube, add 1 mL Isolation Buffer, mix, centrifuge at 10,000 × g for 1 min, and discard the supernatant. Repeat the wash 1-2 times and finally resuspend the beads in 20 μL Isolation Buffer.

4. Transfer 100 μL cell suspension (1 × 10⁷ cells) to the bottom of a sterile centrifuge tube. Add 20 μL of pre-washed beads, mix thoroughly, and incubate at 4°C for 15 min.

Note: Scale the bead volume proportionally for larger cell numbers. For fewer than 1 × 10⁷ cells, adjust the suspension volume to 100 μL and add 20 μL beads.

5. After incubation, add 2.5 mL Isolation Buffer and mix gently.

6. Place the tube on a magnetic separation rack and allow it to stand for 5 min.

7. While keeping the tube on the magnetic rack, carefully transfer the supernatant to a new sterile centrifuge tube. The supernatant contains the CD3⁺-depleted cell population.

8. To increase depletion efficiency, the collected supernatant may be subjected to a second round of bead treatment by repeating Steps 3-7.

9. Centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cells. Wash and resuspend the cells as required for downstream experiments.