My collegues are currently working with a non-adherent human monocyte cell line - how can we separate the dead cells for passage?
Thank you
You can use a Percoll or Ficoll density gradient. Transfer the cell of into a tube and centrifuge for 3-5 min with 1200 rpm in a swing out rotor. Take the supernatant away and resuspend the cells in e.g. 5 ml PBS. Then just layer your cells on top of 5 ml Ficoll-Paque (or Percoll) in a 10 ml tube and centrifuge for 20 min with 3000 rpm WITHOUT break (if you have more than 20 million cells use 10 ml Ficoll and a 50 ml tube, you may also scale down the approach to 2.5ml in a FACScan tube or even to 500 µl in a 1.5 or 2 ml Eppendorf tube). You can also apply your hybridoma supernatant directly to the Ficoll. Dead cell will end up in the bottom of the tube due to their shrinking and increased granularity = higher density. The layer of living cells can be harvested,washed with PBS and plated or analyzed further.
I am think the same answer that Andre Fernandes, try to do it.
I've used this gradient centrifugation several times to recover precious cell non-adherent cultures. From my experience, it worked each and every time, though once a dying cell culture you will only prolong a bit your "agony".
- Badges
- Science method