Human CD3⁺ T Cells Negative Selection Kit

Recommended Buffer (Not Provided)

Magnetic Bead Pretreatment

Thoroughly resuspend the Streptavidin Magnetic Beads. Transfer 20 μL of beads into 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 the wash 1-2 times. Finally, resuspend the beads in 20 μL of Isolation Buffer.

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

Human Peripheral Blood Mononuclear Cell (PBMC) Preparation

Isolate PBMCs from human peripheral blood by Ficoll density-gradient centrifugation. Wash the cells with PBS and centrifuge. Resuspend the PBMCs in Isolation Buffer and adjust the cell density to 1 × 10⁸ cells/mL.

Cell Isolation

1. Antibody Labeling: Transfer 100 μL of cell suspension containing 1 × 10⁷ cells to the bottom of a flow tube. Add 2 μL of Biotin-Antibody Mix, mix well, and incubate at 4°C for 10-15 min.

Notes: a. Transfer the cell suspension directly to the bottom of the tube and avoid dispensing it along the tube wall;
b. For larger cell numbers, increase the amount of Biotin-Antibody Mix proportionally.

2. Cell Washing: After antibody incubation, wash the cells once with 10 volumes of Isolation Buffer. Centrifuge at 500 × g for 5 min and discard the supernatant.

3. Magnetic Bead Labeling: Resuspend the cells in 100 μL of Isolation Buffer. Add 10-20 μL of pre-washed magnetic beads, mix well, and incubate at 4°C for 10 min.

Note: For 5 × 10⁷ cells, adjust the cell-suspension volume to 500 μL and proportionally add 10 μL of Biotin-Antibody Mix and 50-100 μL of beads. When sorting fewer than 1 × 10⁷ cells, adjust the cell-suspension volume to 100 μL.

4. Washing: After incubation, add 2.5 mL of Isolation Buffer to the flow tube and gently pipette to mix. Avoid vigorous shaking or repeated inversion.

5. Magnetic Separation: Place the flow tube containing the cells on a magnetic separator for 5 min.

6. Cell Collection: Keep the flow tube on the magnetic separator and carefully transfer the cell suspension in the supernatant to a new sterile centrifuge tube. The collected suspension contains untouched CD3⁺ T cells. Centrifuge at 500 × g for 5 min, discard the supernatant, and resuspend the cells as required for downstream experiments.

Note: To further increase purity, centrifuge the collected cells, resuspend them in 100 μL of Isolation Buffer, and perform a second round of magnetic separation using the same procedure.