Apr 20, 2024

Public workspaceCD34+ isolation from human bone marrow V.2

DOI
dx.doi.org/10.17504/protocols.io.36wgq4p53vk5/v2
  • Mohsen Khosravi-Maharlooei1,
  • Markus Holzl1,
  • Austin Chen1,
  • Megan Sykes1
  • 1Columbia Center for Translational Immunology, Columbia University, New York
Sandy Beshir
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgq4p53vk5/v2
Protocol Citation: Mohsen Khosravi-Maharlooei, Markus Holzl, Austin Chen, Megan Sykes 2024. CD34+ isolation from human bone marrow. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq4p53vk5/v2Version created by Sandy Beshir
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2024
Last Modified: April 20, 2024
Protocol Integer ID: 98527
Funders Acknowledgement:
NIH
Grant ID: U01 DK123559
Abstract
This protocol details the steps for isolating CD34+ cells from human bone marrow. The CD34+ cells isolated from this protocol can be used for generating humanized mice through reconstitution of immune cells via IV injection after bone marrow ablation. These cells can also be used for mixed lymphocyte reaction experiments.



Note
Corresponding Authors

Mohsen Khosravi-Maharlooei
Email: mkm2182@cumc.columbia.edu

Austin Chen
Email: ac4274@cumc.columbia.edu
Tel: 425-283-6900

Materials
Required material


Required Buffers

  • BM Medium (Amount500 mL Media 199, Amount5 mL Hepes, Amount5 mL DNAse, Amount40 µL Gentamycin)
  • MACS buffer (Amount500 mL PBS, Amount5 g BSA, Amount2 mL EDTA, sterile filtrated and degassed)
  • Cryomedium (Amount90 mL PBS, Amount10 mL FBS, Amount10 mL DMSO)









Before start
Human bone marrow is a rich source for CD34+ hematopoietic stem cells. CD34+ cells can be easily isolated and further processed.

Transfer the content of the collection bag into a sterile flask
Add Amount250 mL BM Medium to the bag and rinse it thoroughly

Transfer the content of the collection bag into the sterile flask
Layer Amount35 mL of the suspension over Amount15 mL of Histopaque

Centrifuge the tubes for 30 minutes Amount500 g without brake at RT

Collect the leukocyte ring in Amount50 mL Falcons and fill up with BM medium

Wash once by centrifuging 6 minutes Amount500 g

Resuspend the cells in MACS buffer and count (take Amount50 µL for FACS confirmation = PRE)

Wash down again and resuspend the pellet according to the protocol (130-046-702 MACS Human CD34+ kit)
Add Amount300 µL of MACS buffer per 10^8 cells

Add Amount100 µL of FcR-B reagent per 10^8 cells, mix it and incubate in fridge for 15 minutes

Add Amount100 µL of CD34 beads per 10^8 cells to the suspension and mix and let it sit for 30 min (fridge)

Fill up with Amount50 mL MACS buffer and strain through a blue strainer (40 uM)

Wash cells (Amount500 g 6 min) and resuspend in MACS buffer 500 uL/200,000,000 cells. If you have more cells, increase volume accordingly. E.g 3 *10^9 cells = 7,5mL. Aliquot this volume to more than one (with Amount3 mL prerinsed) MACS column.

Wash with buffer Amount3 mL 3 times and keep negative fraction (Take Amount50 µL for FCM = POST neg)

Put the column out of the magnet and push out positive fraction with Amount5 mL Buffer and the plunger

Collect the positive fractions. (Take Amount50 µL for FCM = Post pos)

Process the cells as desired (injection in mouse or cryopreservation)
Check the puritiy with FACS