It is fairly simple to separate dead cells and living cells via Percoll or Ficoll density gradient centrifugation (Ficoll-Paque is cheaper!). 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 (hybridoma, PBMCs, tumor cell lines etc.) 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.

Simply method have I do in my lab always is change medium between 2-3 hours than 12 hours after passage.. checked percentage cell viability and it's possible to make limiting dilution from cell suspension.

Hope will help you..

The best method is Flow cytometry cell sorting

Thanks a lot. I have decided to exchange culture medium every 2 days. My hybridioma line proliferates well so there should be no problem to get rid of the most of dead cells from the suspension before we will freeze them or transfer them to bioreactor bottle. I'm sceptic about flow cytometry because we use antibiotic-free medium.

We use the 3 minute technique: take the plates out of the incubator, check for wells that have non viable cells, gently pippete using the apropriate pippete tip, in circular movements. Let it rest 3 minutes (viable cells will settle down, while dead cells and debri will take longer) then aspirate about half or 2/3 of the culture medium. Pour fresh medium on the wells and take it back to the incubator.

Thanks Andrei, this is really clever way!

No problem mate, just be carefull not to aspirate way too much, or you will lose some precious cells!

Andrei, this seems to contradict Bernhard's recommendation above, who says that dead cells have a HIGHER density than viable cells, whereas your method implies dead cells have a LOWER density than viable cells. Can anyone comment on this?

Hi Paul Spencer Hole!

I think I can comment on your question. Bernhard B. Singer suggestion involves a high density medium while Andrei Moroz uses regular medium. A dead cell with a compromised cell membrane will let the surrounding medium immerse the cell (which is the working principle for most viability dyes). When the surrounding medium enters the dead cell it will alter the mechanical properties such as density. So a dead cell in ficoll will have higher density than a dead cell in regular medium.

If you are interested, we recently showed that the density and compressibility of dead cells, with a compromised cell membrane, is a function of the material properties of the surrounding medium.

Article Acoustic separation of living and dead cells using high density medium