Nov 10, 2023
Isolation of Nucleated Cells from Bone Marrow Aspirate
- Steven B. Wells1,
- Peter A. Szabo2,
- Nora Lam2,3
- 1Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 2Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 3Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032
- Columbia
Protocol Citation: Steven B. Wells, Peter A. Szabo, Nora Lam 2023. Isolation of Nucleated Cells from Bone Marrow Aspirate. protocols.io https://dx.doi.org/10.17504/protocols.io.bwrupd6w
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: July 21, 2021
Last Modified: November 10, 2023
Protocol Integer ID: 51732
Keywords: Bone marrow, CD45, Lymphocytes, Myeloid, Isolation, Density gradient, Ficoll, Immune, 10x, scRNAseq, Flow cytometry, WBC, Leukocyte, Single cell suspension, T cell, Progenitor, bone marrow aspirate this protocol, isolation of nucleated cell, human bone marrow aspirate, progenitors from human bone marrow aspirate, bone marrow aspirate, cell suspensions across sample, nucleated cell, single cell suspension, cell suspension, cell, method for the isolation, density centrifugation, dilution factor for density centrifugation,
Abstract
This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human bone marrow aspirate. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.
Attachments
dzhkbk587.pdf
85KB
Materials
Materials:
- DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
- Penicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
- Thermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
- Gibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
- Gibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
- UltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
- Ficoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
- Mr. Frosty™ Freezing ContainerFisher ScientificCatalog #5100-0001
- CryoStor CS10 100MLFisher ScientificCatalog #NC9930384
- Corning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
- 5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
- Solution 13 AO – DAPIChemometecCatalog #910-3013
- NC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003
Equipment:
- Centrifuge
- Cell Counter - NC-3000
Troubleshooting
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a 500 mL bottle of IMDM by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| IMDM | 500 | - | - | |
| Penicillin-Streptomycin-Glutamine | 5 | 100X | 1X | |
| FBS | 50 | 100% | 10% |
Table 1.
*Final Concentration is approximate.
Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| DPBS | 500 | - | - | |
| FBS | 25 | 100% | 5% | |
| EDTA | 1 | 0.5M | 1mM |
Table 2.
*Final Concentration is approximate.
Preparation of Bone Marrow
Record the total volume of bone marrow to be processed.
__________mL.
Divide the bone marrow into 10 mL aliquots and distribute to separate 50 mL tubes.
Dilute the bone marrow using 4 volumes or 40 mL of DPBS-FBS-EDTA Solution; invert to mix.
Note
NOTE: This is the optimum dilution to maximize cell recovery.
Ficoll-Paque
40m
Layer the bone marrow/ DPBS-FBS-EDTA Solution mixture from the 50 mL tubes 25 mL at a time in separate 50 mL tubes on top of 15 mL of Ficoll-Paque Media PLUS.
Note
NOTE: For any remaining volume, add DPBS-FBS-EDTA Solution to bring the volume to 25 mL , and layer as described in this step.
Spin for 00:20:00 , 1200 x g at 20 °C with 4 acceleration and 0 brake, evenly distribute the tubes across the entire rotor to prevent wobbling (use all four buckets if possible as opposed to just two).
20m
Remove the mononuclear cell layer from each tube with a transfer pipet to 50 mL tubes - mononuclear layers may be combined at this step to reduce the number of tubes to spin. Add cold DPBS-FBS-EDTA Solution to a final volume of 50 mL and centrifuge the cell suspensions for 00:10:00 at 400 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 00:10:00 at 120 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.
Cell Count
Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: __________cells/mL, __________% viable
final volume: __________mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: __________
Freeze-down and QC
(Optional QC) Aliquot 2 x 106 cells to a 5mL Falcon tube and place on ice for subsequent flow cytometric analysis.
Aliquot cells for analysis or experimentation, and then freeze down remaining in up to 3 x 107 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a -80 °C freezer (1 mL -1.5 mL aliquots, round down to the nearest 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.