The protocol is derived from the WHO/IARC guideline “Common Minimal Technical Standards and Protocols”. The purpose of the document is to provide a framework for the development and coordination of Biological Resource Centers.
White blood cells collected in EDTA and ACD tubes, can be used for DNA extraction and the creation of cell lines.
Instead of a separation based on Ficoll, a Percoll separation can be used alternatively.
- Cell culture media
- RPMI 1640
- DMSO , 10%
- Fetal calf serum , (FCS)
- Transfer the remaining blood from the plasma spin to a labeled 50ml tube containing 10ml RPMI 1640.
- After alcohol swabbing the lid of this tube, aliquot 3ml Ficoll into each of two clearly labelled 15ml tubes.
- Carefully layer 9ml diluted blood onto each tube of Ficoll. Treat gently, do not mix, but spin as soon as possible.
- Pour off the supernatant into a waste container containing chlorine bleach.
- Add 3ml of cold freezing mix (10% DMSO, 20% FCS, RPMI 1640) and resuspend.
- Dispense the white blood cells into 3 x 1ml labeled cryovials, which have been sitting on ice.
- Place vials in a rate-limiting freezer as to cryopreserve cells in conditions that maintain cell viability.
- This should be done as soon as possible as DMSO is toxic at room temperature.
- INFO: Transfer on a weekly basis to liquid nitrogen tanks.
- Spin at 450xg for 30 minutes, when centrifuging, do not use brake.
- Remove most of the top layer (RPMI 1640) using a 1ml Eppendorf tip and discard (≈3-4ml) into waste container containing chlorine bleach. Collect white blood cells with the same Eppendorf tip using a swirling motion to ‘vacuum up’ white blood cells.
- Do not take too much Ficoll (third layer), as it is toxic to the cells.
- Place the white blood cells in a labeled 15ml tube containing 10ml RPMI.
- Spin at 450xg for 10 minutes.