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I am trying to knock-out genes in BV-2 cell line. However, the majority of protocols require cell subcloning and expanding. I tried to subcone these cells and grow clones in DMEM+10% heat-inactivated FBS+1% PS (usual media I am using)+Insulin-Transferrin-Selenium. My cells did not grow and eventually died. Maybe there are supplements that I can add to the media to promote cell growth from one cell? I am considering using conditioned media from the same BV-2 cell culture after culturing them for a few days. Should it work?

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  • $\begingroup$ Welcome to the Biology Stack Exchange. Please take a tour and visit the help center for more information about the site and what we do here. This isn't a simple question and answer site, so things are a little different to more conventional forums. $\endgroup$
    – bob1
    Jan 24, 2023 at 18:37
  • $\begingroup$ We would need a few more details on how you go about your subcloning to troubleshoot why it isn't working. $\endgroup$
    – bob1
    Jan 24, 2023 at 18:38
  • $\begingroup$ As always, consulting reference materials is a good idea: sigmaaldrich.com/US/en/technical-documents/technical-article/… $\endgroup$ Jan 24, 2023 at 20:44
  • $\begingroup$ I am growing these cells routinely in the lab, and they grow fine with usual media (cell thawing is always successful, they are not contaminated etc.). When I do subcloning i calculate cells so there is one cell in 100ul media. And then i seed them in 96-wp. After they attach i search wells where is only one cell in the well (subclone). And the next step would be expanding the subclones. I read articles where similar things were done and did not find any specific requirements for successful subcloning. $\endgroup$
    – Monika1223
    Jan 25, 2023 at 5:31
  • $\begingroup$ Are your knockouts lethal? $\endgroup$
    – MattDMo
    Jan 26, 2023 at 15:37

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