Human CD34+ Cell Enrichment Kit (negative selection)

Mouse Cells

Human Cells

1.High Activity: After isolation, cell function remains intact with no abnormal activation, and no antibody or magnetic bead labeling.

2.Easy Operation: No separation column is required; target cell isolation can be achieved using a magnetic rack.

3.Fast Processing: CD34+ cell enrichment can be completed in as little as 30 minutes.

4.High Enrichment: A single enrichment step achieves a 10-fold concentration, while two steps achieve a 30-fold concentration.

Suitable for enriching CD34+ cells from umbilical cord blood mononuclear cells (CBMC) or peripheral blood mononuclear cells (PBMC).

1.To prepare the human cord blood mononuclear cells (CBMC) or human peripheral blood mononuclear cell (PBMC) suspension: Isolate mononuclear cells from human cord blood or peripheral blood using Ficoll density gradient centrifugation. Wash the cells with PBS and then centrifuge. Resuspend the CBMC or PBMC in the isolation buffer, and adjust the cell concentration to 1×10^8 cells/mL.

Note: Recommended isolation buffer: a. PBS（2 mM EDTA and 2% FBS）; b. PBS（2 mM EDTA and 0.5% BSA）. The buffer should be pre-filtered using a 0.22 μm membrane for sterilization.

2.Transfer 100 μL of the cell suspension (1×10^7 cells) to the bottom of a sterile tube. Add 2 μL of Biotin-Antibody Mix, mix thoroughly, and incubate at 4°C for 10 minutes.

Note: a. Transfer cell suspension directly to the bottom of the tube, avoiding adding along the wall of the tube. b. If a larger quantity of cells requires sorting, proportionally increase the volume of Biotin-Antibody Mix used.

3.To prepare the magnetic beads: Vortex to resuspend the beads. Transfer the required amount of beads to a 1.5 mL centrifuge tube. Add 1 mL of isolation buffer, and centrifuge at 10,000 g for 1 minute. Discard the supernatant and repeat the washing step once.

Note: The volume of isolation buffer used for beads resuspension should be equal to the initial volume of beads that was aspirated. E.g., if 20 μL of beads are used for washing, resuspend in 20 μL of isolation buffer.

4.After cell-antibody incubation, add 20 μL of pre-treated Streptavidin Magnetic Beads to the tube, mix well and incubate at 4°C for 10 min.

Note: If a larger number of cells need to be sorted, the amount of Streptavidin Magnetic Beads can be increased proportionally. For sorting 5×10^7 cells, add 10 μL of Biotin-Antibody Mix and 100 μL of beads to 500 μL of cell suspension. If sorting less than 1×10^7 cells, adjust the volume of the cell suspension to 100 μL and add 2 μL of Biotin-Antibody Mix along with 20 μL of beads.

5.After incubation, add 2.5 mL of isolation buffer to the tube and mix 5 times gently (avoid vigorous shaking or up-and-down mixing).

6.Place the tube in the magnetic separator for 5 minute.

7.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 human CD34+ cells. Centrifuge at 500 g for 5 minutes, discard the supernatant and collect the cells.

Note: To further improve the enrichment of CD34+ cells, steps 2-7 can be repeated once.

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

Store at 4℃ for 2 years

1.Avoid freezing. Store beads in the solution to prevent drying.

2.Before removing beads from the tube, ensure they are evenly suspended by gentle shaking. Handle gently to prevent the bubbles.

3.It is recommended to use high-quality pipette tips and centrifuge tubes to avoid loss of beads due to adhesion.

4.This product is for scientific research use by professionals only and must not be used for clinical diagnosis or treatment, food or drug applications, and must not be stored in residential areas.

5.For your safety and health, wear lab coats and disposable gloves during operation.

• C0066 Instruction Mannual(123 KB)