I know gradient centrifuge is a good way to separate live cells from dead cells and debris, but I also notice that many people do a simplified "low speed centrifuge" to remove dead cells. It is logical and plausible that dead cells are smaller and lighter compared to live cells, thus they should come down to the bottom latter during the centrifuge step. I once tried to do this on mouse B lymphocyte and it works. However, it is not always the case: I tried to remove dead cells in hybridoma medium, with 250g for 3min, and the pellet contained nearly 90% dead cells. I know the ratio of dead/live cell and it is impossible. It means my live cells remained in the supernatant...
So what's wrong with this centrifuge? Have anyone used low speed centrifuge?
PS. Since I don't have enough live cells left after the centrifuge, I cannot do a dilution passage. I don't know what effect will such big number of dead cells have on my live cells. Any suggestion to keep them grow?
Thanks
Just a quick shot:
Dead cell removal: Dead cells are removed during cell isolation using Ficoll-Paque PLUS
(found in: http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/Isolation%20of%20Mononuclear%20Cells.pdf )
NB: edited 2017-01-28: please try also [to be found by search function in RG = ] 'Search' 'Questions' for: e. g.: | live dead cells separation | (creates the URL:
https://www.researchgate.net/search.Search.html?type=question&query=live+dead+cells+separation ) and find similar questions and replies, e.g. esp. https://www.researchgate.net/post/Has_anyone_used_Histopaque_1077_to_separate_live_and_dead_culture_cells
Hi Zhitao, if you are growing your hybridoma cells in serum-containing medium, then you could try layering the cells on 100% serum and centrifuge at low speed. The serum creates a density interface - live cells will sediment faster than dead cells and cell debris. This is a gentle technique. You need to be mindful though that there is an increased risk that antibody production will be switched-off in stressed hybridoma cells. Good luck.
Regards, Rosemary
I heard the other way around but I am not sure 100%. Centrifuge at 800-1000xg for 3-5 minutes, so that nearly almost dead cells will burst and will be in the supernatant. might not be logical but if anyone can explain better, I would be appreciated. I always tried to use 300-400xg to remove dead cells before.