I am using MCF10A cell lines. I see different sensitivity to Blasticidin of my control cells (not infected with lentivirus) vs cells infected with lentivirus. I thought this is a good thing as My infected cells do not die as fast as the control cells but unfortunately the infected cells die after 2-3 passages (Even after splitting the cells in 1:2 ratio).
One area I am looking into is maybe the virus is not being made in the first place and just the plasmids are getting carried over. That is why we get resistance initially and not after passaging. I have ordered a viral titer determination kit and also doing the experiment with lenti GFP (use a fluorescence marker) to determine if the plasmids are working correctly.
But I also think that there might be some problem with Blasticidin as a drug. I would be glad if people can share their experiences. Thank you!
Two things happened with while facing the same issue but with different type of cells:
(1) lentivirus preparation was contaminated with mycoplasma. Got red of it by extensive treatment of cells with anti-mycoplasma agent.
(2) improper adjustment after thawing of cells (DMSO accumulative killing effect). Got red of it by warming cells 37C + put it in 10ml media + Centrifuge + re-suspend the cellular pellet in 15ml new media + put in T75 flask.
Hope this may help.
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