Freezing and
Thawing PBMC's
Contact: Ometa Herman
|
Materials |
Qty |
Location |
Order Info |
|
|
B-008 Cold room |
Irvine 9513 (w/o glutamine) See Herzenberg Lab Media Preparation for details |
|
|
FCS |
|
|
|
|
DMSO |
|
|
|
|
Cryovials |
|
|
|
|
Ethidium Bromide/ Acridine Orange |
|
B-013 Fridge #7 |
|
Freezing
Final PBMC conc: 5x10^6 c/ml
Final DMSO conc: 10%
Aliquot Size: 0.5 ml (2.5x10^6 c)
1. Spin down freshly isolated and depleted PBMC's
2. Resuspend in RPMI* containing 20% FCS (10x10^6 c/ml)
3. Place on ice and let cool
4. Dropwise add an equal volume of ice cold RPMI/20% DMSO (Final concentration is 10% DMSO)
5. Aliquot into cryovials on ice (0.5 ml)
6. Let stand 30 min on ice
7. Freeze down at 1 C/min or use isopropyl freezing chamber in –80 freezer.
8. Store in LN2 after 1 day.
Note: For more sensitive cells these conditions may prove too harsh. Alternatively, you can resuspend cells in FCS, and then dropwise add an equal volume of FCS containing 20% DMSO, aliquot into vials at room temperature, and place directly into –80 freezer in Styrofoam box or isopropyl freezing chamber. Store in LN2 the next day.
Thawing
1. Thaw cells by placing cryovial in 37 C bath
2. When only a small piece of ice remains, add 1 ml of RPMI/10%FCS dropwise
3. Transfer into a 15 ml conical and add 10 ml of RPMI/10%FCS
4. Spin down (1000 rpm, 4') and resuspend in 1 ml RPMI/10%FCS
5. Test viability by adding an equal volume of EB/AO to a small aliquot of cells