TITLE: Isolation of Nucleated Cells from Whole Blood
AUTHORS: Steven B. Wells, Peter A. Szabo, Nora Lam

[DESCRIPTION]
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.

[STEPS]
SECTION: Preparing Mediums and Buffers
1. Create the following IMDM-FBS-PSQ Media in a 500mL bottle of IMDM by using the table below: 
      Component  Volume (mL)  Starting Conc.   Final Conc.*    IMDM  500  -  -    Penicillin-Streptomycin-Glutamine  5  100X  1X    FBS  50  100%  10%  
 
SECTION: Preparing Mediums and Buffers
2. Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS by using the table below: 
      Component  Volume (mL)  Starting Conc.   Final Conc.*    DPBS  500  -  -    FBS  25  100%  5%    EDTA  1  0.5M  1mM  
 
SECTION: Preparation of Blood
3. Record the total volume of whole blood to be processed.
__________mL
SECTION: Preparation of Blood
4. Spin the whole blood 400 x g for 10 min in the anti-coagulant tubes, remove the plasma layer, and distribute to cryovials – up to 2 mL/vial.
Record the total volume of plasma: __________ mL and the number of vials: __________.
SECTION: Preparation of Blood
5. Replace the plasma volume removed from the whole blood with DPBS-FBS-EDTA Solution.
SECTION: Preparation of Blood
6. Divide the whole blood into 10 mL aliquots and distribute to separate 50 mL tubes.

SECTION: Preparation of Blood
7. Dilute the whole blood using 4 volumes or 40 mL DPBS-FBS-EDTA Solution; invert to mix.
 


SECTION: Ficoll-Paque
8. Layer the blood/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.
 

SECTION: Ficoll-Paque
9. Spin for 20 min, 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).

SECTION: Ficoll-Paque
10. 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 10 min at 400 x g, 4 °C.

SECTION: Ficoll-Paque
11. Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 10 min at 120 x g, 4 °C.

SECTION: Ficoll-Paque
12. Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.

SECTION: Cell Count
13. 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: __________
SECTION: Freeze-down and QC
14. (Optional QC) Aliquot 2 x 106 cells to a 5 mL Falcon tube and place on ice for subsequent flow cytometric analysis.

SECTION: Freeze-down and QC
15. 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 (1 mL-1.5 mL aliquots, round down to the nearest 20 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.