Author Stephanie Swift
Date 3rd June 2015
Version 1
SOP # H001
Reageants
Ficoll (or Leukosep)
PBS
DMSO
Human Serum Albumin (HSA)
RPMI media
Objective: This protocol addresses the processing of freshly-drawn human blood into plasma and PBMC samples.
H001 - Processing Human Blood
A. Preparation of Reagents
1. Preparation of 12.5% HSA: a) Split HSA into 2 x 50 mL conical tubes (2.5 g each) and pipet 20 ml RPMI (no additives) into each tube. b) Knock on table to wet HSA powder and place overnight at 4 o C or two (2) hours at room temperature (RT). Note: Do not agitate excessively as the solution becomes foamy. c) Once dissolved, filter through 0.2µm filter into a sterile tube in the BSC. d) Store filtered HSA at 4 o C.
2. Preparation of 2x Freezing Media: a) Slowly drip 2.5 mLs DMSO into 10 mLs 12.5% HSA while gently mixing (swirling motion).
Note: Clumps will disappear with sufficient gentle mixing. Do not pipet DMSO down side of polypropylene tube; clumps are forms that will not dissolve.
B. Processing Human Blood
1. Measure the total volume of blood across all tubes collected for each patient, and make a note in your labbook.
2. Centrifuge blood collection tubes for 10 minutes at 1500RPM to separate plasma.
3. Remove yellow upper layer from each tube (if plasma required- aliquot 3 x 1ml to cryovials and store at -20 o C).
4. Using a 10 ml pipet, transfer blood sample into 50 ml tubes (maximum of 10 mLs
/ tube).
5. Add an equal volume of PBS (up to 10ml maximum) to blood and mix gently with pipet.
6. Slowly pipet equal volume of Ficoll under diluted blood so Ficoll layer forms below diluted blood layer (10ml blood + 10ml PBS + 20ml Ficoll). When done properly two (2) distinct layers should be observed.
This protocol is shared by the Stojdl Lab under a creative commons license.

7. Centrifuge tubes for 25 minutes ( no brake) at 1500RPM. A thin white interface should be observed between the top and middle layer after centrifugation.
NOTE: This is the PBMC population to be isolated.
8. Draw off top plasma layer using a 10ml pipette, leaving at least 5ml above the interface (this helps to remove platelet contamination). Be careful not to disturb the interface. Gently aspirate interface using 10ml pipette into a new 50ml tube.
Do not combine interface from multiple tubes; do a 1:1 transfer.
9. Pour PBS into the tube to 50ml. Mix by inversion. Centrifuge 10 minutes at
1500RPM at room temperature.
10. Pour supernatant into waste bucket and gently resuspend cells, combining tubes at this point in ~20 ml PBS.
Note: if pellet is small add 10 – 15 ml PBS instead of 20 ml.
11. Remove 20µl cell suspension and add equal amount of Trypan Blue in a nonsterile 96-well plate or an eppendorf tube, and gently mix. Count cells using a haemacytometer (only mononuclear cells (large, grayish cells), not RBCs (small, round, shiny cells)).
N.B. If you are proceeding immediately on to stain cells, centrifuge samples for 5 minutes at 1500RPM at room temperature and resuspend at 2x10^7 cells/ml and plate 100ul per well. Proceed with protocol H002
(tetramer/immunophenotyping) or H003 (ICS). Otherwise, continue with step 11 to freeze down cells.
12. Centrifuge samples for 5 minutes at 1500RPM at room temperature.
13. Calculate the number of cryovials required to achieve 5 -10 x 10 6 cells / ml in each cryovial.
14. Label each vial with:
- Patient Identification
- Date Processed
- Number of Cells / Vial
- " PBMC’s"
- Technician Initials
15. Cool cryovials in freezing container for a few minutes.
16. Resuspend the pellet in cold 12.5% HSA (see section A, above). Add an equal volume of freezing media so that the final concentration is 5 – 10 x 10 6 cells / mL
(i.e. 4 ml total = 2 ml HSA + 2 ml freezing media), gently mix well using a swirl motion.
17. Transfer 1 ml cell suspension into each labeled cryovial.
18. Store freezing container overnight (24 hours) at -80 o C.
IMPORTANT NOTE: Freezing container must remain immobile for the first four (4) hours after storage at -80 o C. If it is moved or bumped, the freezing process is disrupted and the cells may be damaged.
19. Transfer tubes into liquid nitrogen tank.
20. Add 5 – 10ml bleach to waste bucket and let sit for 30 minutes before dumping down sink.
Protocol originally modified from the Bramson lab, McMaster University, Hamilton,
Canada
This protocol is shared by the Stojdl Lab under a creative commons license.