Apr 19, 2024
Version 2
CD34+ isolation from human bone marrow V.2
- Mohsen Khosravi-Maharlooei1,
- Markus Holzl1,
- Austin Chen1,
- Megan Sykes1
- 1Columbia Center for Translational Immunology, Columbia University, New York
- Human Islet Research Network
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 19, 2024
Last Modified: April 19, 2024
Protocol Integer ID: 98527
Keywords: isolating cd34, isolation from human bone marrow, cells from human bone marrow, cd34, immune cells via iv injection, human bone marrow, immune cell, bone marrow ablation, mixed lymphocyte reaction experiment, bone marrow, humanized mice, cell, mice through reconstitution, iv injection
Funders Acknowledgements:
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: [email protected]
Austin Chen
Email: [email protected]
Tel: 425-283-6900
Materials
Required material
- MACS buffer(degassed)
- Sterile flask for BM rinse
- Histopaque
- Sterile pipetts
- MACS supply
- antibodies
Required Buffers
- BM Medium (500 mL Media 199, 5 mL Hepes, 5 mL DNAse, 40 µL Gentamycin)
- MACS buffer (500 mL PBS, 5 g BSA, 2 mL EDTA, sterile filtrated and degassed)
- Cryomedium (90 mL PBS, 10 mL FBS, 10 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 250 mL BM Medium to the bag and rinse it thoroughly
Transfer the content of the collection bag into the sterile flask
Layer 35 mL of the suspension over 15 mL of Histopaque
Centrifuge the tubes for 30 minutes 500 g without brake at RT
Collect the leukocyte ring in 50 mL Falcons and fill up with BM medium
Wash once by centrifuging 6 minutes 500 g
Resuspend the cells in MACS buffer and count (take 50 µL for FACS confirmation = PRE)
Wash down again and resuspend the pellet according to the protocol (130-046-702 MACS Human CD34+ kit)
Add 300 µL of MACS buffer per 10^8 cells
Add 100 µL of FcR-B reagent per 10^8 cells, mix it and incubate in fridge for 15 minutes
Add 100 µL of CD34 beads per 10^8 cells to the suspension and mix and let it sit for 30 min (fridge)
Fill up with 50 mL MACS buffer and strain through a blue strainer (40 uM)
Wash cells (500 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 3 mL prerinsed) MACS column.
Wash with buffer 3 mL 3 times and keep negative fraction (Take 50 µL for FCM = POST neg)
Put the column out of the magnet and push out positive fraction with 5 mL Buffer and the plunger
Collect the positive fractions. (Take 50 µL for FCM = Post pos)
Process the cells as desired (injection in mouse or cryopreservation)
Check the puritiy with FACS