Mouse Pan B Cell Negative 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 pan B cells from mouse spleen, lymph nodes, bone marrow, or peritoneal lavage

1. Prepare a single-cell suspension from mouse spleen, lymph nodes, bone marrow, or peritoneal lavage. Remove red blood cells as appropriate, resuspend the cells in PBS, filter through a 70 μm cell strainer, and count the cells. Centrifuge at 500 × g for 5 min and discard the supernatant.

Note: This step removes cell clumps or tissue debris that may affect separation purity.

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

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

Note: a. Add the cell suspension directly to the bottom of the tube rather than along the tube wall;
b. Scale the amount of Biotin-Antibody Mix proportionally for larger cell numbers.

4. Preparation of Streptavidin Magnetic Beads: thoroughly resuspend the beads and transfer 20 μL 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.

Note: The final volume of Isolation Buffer used to resuspend the beads should equal the initial bead volume.

5. After antibody incubation, add 20 μL of pre-washed Streptavidin Magnetic Beads, mix thoroughly, and incubate at 4°C for 10 min.

Note: Scale reagent amounts proportionally for larger cell numbers. For fewer than 1 × 10⁷ cells, adjust the cell suspension volume to 100 μL and use 2 μL Biotin-Antibody Mix and 20 μL beads.

6. Add 2.5 mL Isolation Buffer and mix gently, avoiding vigorous vortexing or repeated pipetting.

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

8. While the tube remains on the magnetic rack, carefully collect the cell suspension into a new sterile centrifuge tube. The suspension contains enriched mouse pan B cells. Centrifuge at 300 × g for 5 min, discard the supernatant, and collect the cells.

Note: Avoid touching the beads with the pipette tip; the cell suspension may also be collected by pouring.

9. Wash the cells as required and resuspend them in an appropriate buffer or culture medium. The isolated cells may be used for downstream molecular or cellular experiments.