Isolation of Nucleated Cells from Whole Blood
Steven B. Wells, Peter A. Szabo, Nora Lam
Blood
CD45
Lymphocytes
Myeloid
Isolation
Density gradient
Ficoll
Immune
10x
scRNAseq
Flow cytometry
WBC
Leukocyte
Single cell suspension
T cell
Abstract
This protocol describes a method for the isolation of pan-lymphocytes and pan-myeloid cells from human whole blood. 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
Steps
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a 500mL 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 Blood
Record the total volume of whole blood to be processed.
__________mL
Spin the whole blood 400x g,0h 0m 0s for 0h 10m 0s in the anti-coagulant tubes, remove the plasma layer, and distribute to cryovials – up to 2mL/vial.
Record the total volume of plasma: __________ mL and the number of vials: __________.
Replace the plasma volume removed from the whole blood with DPBS-FBS-EDTA Solution.
Divide the whole blood into 10mL aliquots and distribute to separate 50mL tubes.
Dilute the whole blood using 4 volumes or 40mL DPBS-FBS-EDTA Solution; invert to mix.
Ficoll-Paque
Layer the blood/DPBS-FBS-EDTA Solution mixture from the 50mL tubes 25mL at a time in separate 50mL tubes on top of 15mL of Ficoll-Paque Media PLUS.
Spin for 0h 20m 0s, 1200x g,0h 0m 0s 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).
Remove the mononuclear cell layer from each tube with a transfer pipet to 50mL 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 50mL and centrifuge the cell suspensions for 0h 10m 0s at 400x g,0h 0m 0s, 4°C.
Remove the supernatant and re-suspend the cell pellet in 50mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 0h 10m 0s at 120x g,0h 0m 0s, 4°C.
Remove the supernatant and re-suspend the cell pellet in cold 10mL 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 cells in up to 2 x 107 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a -80°C freezer (1mL-1.5mL aliquots, round down to the nearest 20 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.
