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CD34+ Enrichment

Protocol by Ravishankar Madhu

Last Updated: 2023-7-10

Description

This protocol outlines how to enrich White Blood Cells (WBCs) for cells containing the surface cell marker CD34. This enrichment is applicable to both Human and Non-human Primate (NHP) CD34-positive cells.

Reagents and Materials

  • NHP or Human WBCs (usually from Bone Marrow (BM))
  • MACS Buffer
  • Miltenyi LS Magnet Columns
  • Miltenyi Magnet Rack
  • CD34 12.8 Antibody
  • Miltenyi IgM Antibody

Things to Note before starting

Sample Processing

This procedure assumes a starting input of WBCs. This is primarily done by either Lysing Cells with Hemolytic Buffer or via Ficoll Purification. See the following protocols for further explanation:

CD34 enrichments usually require further processing that demands a high degree of sterility. Therefore, it is important to conduct all steps in a TC Hood.

Estimated Timing

This protocol occurs after isolating WBCs from BM, which takes approximately 30 minutes. The enrichment protocol includes two 30-minute incubation steps and several 5-minute spins. On average, the full protocol from BM to enriched CD34 cells will take approximately 3.5-4.0 hours.

Procedure

  1. Resuspend WBCs in 50mL of MACS Buffer.
  2. Using 190uL of MACS Buffer and 10uL of cell suspension, create a 1:10 dilution. 1
  3. Count the cells via the Countess. Calculate the total number of living WBCs.
  4. Save 3 aliquots2 of the 50mL Cell resuspension for Flow Analysis. These 3 aliquots will be unstained, stained with the HSC Panel, and stained with the Lineage Panel.
  5. Spin down the cells at 800 x g for 5 minutes. Aspirate to the pellet.
  6. Resuspend the cell pellet in **1mL of [[MACS Buffer]] per 1e8 WBCs.
  7. Add 12.8 Antibody to the cell suspension to create a final 12.8 concentration of 20ug/mL. 3
  8. Incubate the solution for 30 minutes in a gentle shaker / rocker at 4C.
  9. Wash the solution in 50mL of MACS Buffer. Spin down at 800 x g for 5 minutes. Aspirate to the pellet.
  10. Resuspend pellet in 50mL of MACS Buffer.
  11. Using 190uL of MACS Buffer and 10uL of cell suspension, create a 1:10 dilution. 1
  12. Count the cells via the Countess. Calculate the total number of living WBCs.
  13. Spin down the cells at 800 x g for 5 minutes. Aspirate to the pellet.
  14. Resuspend the cell pellet in **0.9mL of [[MACS Buffer]] per 1e8 of WBCs.
  15. Add 1mL of IgM Antibody per 1e9 of WBCs.
  16. Incubate solution for 30 minutes in a gentle shaker / rocker at 4C.
  17. Wash the solution in 50mL of MACS Buffer. Spin down at 800 x g for 5 minutes. Aspirate to the pellet.
  18. Calculate the number of Miltenyi LS Columns needed for this purification. Generally, use 1 column per 0.6e9 WBCs.
  19. Set up the Miltenyi LS Columns with a Magnet Rack, 70um filter, and 50mL flow through collection tube.
  20. Resuspend cell pellet in 2mL of [[MACS Buffer]] per Miltenyi LS Columns you are going to use.
  21. Wet each column with 2mL of MACS Buffer.
  22. Add 2mL of cell suspension to each column.
  23. Wash each column with 6mL of MACS Buffer.
  24. Wash each column with 7mL of MACS Buffer.
  25. Take the Miltenyi LS Columns off the Magnet Rack. Add 5mL of MACS Buffer and use the provided plunger to elute the cells into a 15mL tube.
  26. Spin down the positive fraction at 800 x g for 5 minutes. Aspirate to the pellet.
  27. Resuspend the cell pellet in an appropriate volume. 4
  28. Count the cells via the Countess.

Next Steps

The purified CD34+ cells should be QCed by flow with the HSC panel. The cells can be further used for the following purposes: - In vitro experiments - RM012 - Freezing Down Living Cells - RM004 - NHP Ex-Vivo Transplant - RM010 - Creating CFCs Plates - RM033 - Creating Cell Pellets

Appendix

Recipe's for Buffers

MACS Buffer

This reagent is used to keep cells undergoing a CD34+ Enrichment stable and happy. This should be stored in either the cold room, or in the TC Room Fridge if being used.

  • 2.5g of Bovine Serum Albumin
  • 2mL of 0.5M EDTA
  • 500mL PBS
  • Sterilized by filtering with a 0.22um Vacuum Filter

Footnotes

  1. The exact dilution can be adjusted depending on your input of cells. If you feel that you do not have enough cells - perform a 1:2 or 1:4 dilution. If you feel that you have a lot of cells - perform a 1:100 dilution. The total volume of your dilution should range from 50uL-200uL. ↩↩

  2. I would recommend an aliquot volume of 100uL ↩

  3. The 12.8 Antibody is located in vials within the -80 Freezer. The stock concentration of the antibody in that tube is written on the tube's exterior. Take care to note the stock concentration as this can change when we get a new batch of 12.8 antibody. ↩

  4. I would recommend 1mL or 5mL depending on the size of the cell pellet. ↩