Thawing PBMCs

2015-09-02

Thawing frozen mononuclear cells ‘ficolled’ from a peripheral blood or bone marrow aspirate isn't as trivial as dealing with most cell lines. However, the following steps can help maximise the recovery of healthy cells.

1. Thaw the cells quickly in a 37 °C waterbath
2. Chill ~10 mL cold thawing buffer (IMDM, 10% FBS, 10 µg/mL DNAse) in a 15ml centrifuge tube
3. Add thawed cells to this in a drop wise fashion with gentle mixing
4. Rest for 10 minutes on ice
5. Centrifuge at 200 × g for 5 minutes
6. Discard supernatant being careful not disturb the pellet
7. Resuspend cell pellet in desired volume (typically 1 mL) of buffer or complete media and count