Nov 09, 2023

Public workspaceIsolation of Nucleated Cells from Bone Marrow Aspirate

DOI
dx.doi.org/10.17504/protocols.io.bwrupd6w
  • 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
Steven B. Wells
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DOI: dx.doi.org/10.17504/protocols.io.bwrupd6w
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 20, 2021
Last Modified: November 09, 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,
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.
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Materials
Materials:

  • ReagentDPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
  • ReagentPenicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
  • ReagentThermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
  • ReagentGibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
  • ReagentGibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
  • ReagentUltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
  • ReagentFicoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
  • ReagentMr. Frosty™ Freezing ContainerFisher ScientificCatalog #5100-0001
  • ReagentCryoStor CS10 100MLFisher ScientificCatalog #NC9930384
  • ReagentCorning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
  • Reagent5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
  • ReagentSolution 13 AO – DAPIChemometecCatalog #910-3013
  • ReagentNC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003

Equipment:

  • Centrifuge
  • Cell Counter - NC-3000











Preparing Mediums and Buffers
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a Amount500 mL bottle of IMDM by using the table below:
ABCD
ComponentVolume (mL)Starting Conc. Final Conc.*
IMDM500--
Penicillin-Streptomycin-Glutamine5100X1X
FBS50100%10%
Table 1.
*Final Concentration is approximate.
Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS by using the table below:
ABCD
ComponentVolume (mL)Starting Conc. Final Conc.*
DPBS500--
FBS25100%5%
EDTA10.5M1mM
Table 2.
*Final Concentration is approximate.
Preparation of Bone Marrow
Preparation of Bone Marrow
Record the total volume of bone marrow to be processed.
__________mL.
Divide the bone marrow into Amount10 mL aliquots and distribute to separate Amount50 mL tubes.

Dilute the bone marrow using 4 volumes or Amount40 mL of DPBS-FBS-EDTA Solution; invert to mix.
Note
NOTE: This is the optimum dilution to maximize cell recovery.


Mix
Ficoll-Paque
Ficoll-Paque
40m
40m
Layer the bone marrow/ DPBS-FBS-EDTA Solution mixture from the Amount50 mL tubes Amount25 mL at a time in separate Amount50 mL tubes on top of Amount15 mL of Ficoll-Paque Media PLUS.
Note
NOTE: For any remaining volume, add DPBS-FBS-EDTA Solution to bring the volume to Amount25 mL , and layer as described in this step.






Spin for Duration00:20:00 , Centrifigation1200 x g at Temperature20 °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
Centrifigation
Remove the mononuclear cell layer from each tube with a transfer pipet to Amount50 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 Amount50 mL and centrifuge the cell suspensions for Duration00:10:00 at Centrifigation400 x g , Temperature4 °C .

10m
Centrifigation
Pipetting
Remove the supernatant and re-suspend the cell pellet in Amount50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for Duration00:10:00 at Centrifigation120 x g , Temperature4 °C .

10m
Centrifigation
Remove the supernatant and re-suspend the cell pellet in cold Amount10 mL IMDM-FBS-PSQ Media.

Cell Count
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
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 Temperature-80 °C freezer (Amount1 mL -Amount1.5 mL aliquots, round down to the nearest 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.