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