PBMC Isolation (Blood Processing) TASC Protocol
1.
Label 2 tubes (serum), 2 tubes (DMSO), 2 tubes (FF – flash frozen) with
-patient number (PT#)
-date
-serum/DMSO/FF
-patient blood draw day #
-#cells (for DMSO and FF)
2.
3.
4.
5.
Keep blood and cells on ice as much as possible.
Spray hood and gloves with 70% EtOH
Fill large beaker with bleach (under sink) and put inside the hood.
Thaw out HI-FBS (also need DMSO)
6.
7.
8.
9.
10.
Add 4mL Ficoll to 15 conical (2x)
Resuspend patient’s blood by pipetting up and down.
Add 8mL of blood slowly to the Ficoll in tube (they should not mix!)
Spin at 1800g, 20 min at 4oC in large centrifuge in TC (Brake OFF).
SERUM: remove 4x1mL serum (top layer) and
a. place 1mL each in two (2) 1.5mL eppendorff.
b. snap freeze in liquid N2
c. store in -80oC.
PBMC ISOLATION:
1. Add 10mL PBS to a 15mL conical
2. Remove white layer below serum (PBMCs) using a Pasteur pipet. Remove slowly moving pipet tip back and forth
across layer while sucking up.
3. Add PBMCs to 10mL PBS.
4. Write down final volume!!!
5. Resuspend PBMCs in PBS by pipetting up and down.
6. Count cells.
a. Dilute 10l sample + 10l Trypan blue
b. Count cells using BioRad Cell Counter
c. Multiply by 2 to account for 1:1 dilution with trypan blue
7.
DMSO:
a.
b.
c.
d.
e.
f.
label 2 cryovials
need at least 500k cells/tube (ideal 1x106 cells/vial)
Put the volume of cells (ex. Enough for 2 1x106 cells in a separate 15 ml conical for DMSO) .
Spin down in large centrifuge 5min, 13000rpm 4oC (BRAKE HIGH!!!)
Remove PBS and freeze in 10%DMSO/serum (can add 1:1 20%DMSO/serum) using cryovials.
Store in cryochamber in -80oC overnight and then transfer to liquid N2 (afterward).
FF pellets
1. Add cells from #6 (note number of cells to a new 15mL conical).
2. Resuspend with 2 ml PBS
3. Split into 2 eppendorffs (1.5mL) at 1ml/vial.
4. Spin 5000rpm, 5min, 4oC in microfuge in cold room.
5. Remove PBS from all pellets.
6. Flash freeze in liquid N2.
7. Put away in -80oC.
PBMC Isolation (Blood Processing) TASC Protocol