I'm dealing HEK 293T cells for protein expression. How is many generations the maximum for passaging? Many thanks.
As above - in theory they can keep forever, however, the longer they are in continuous culture, the more chance you have of contamination and mutation. Best practice is to cryopreserve your own stock form your earliest passages post thawing then return to these every 2-3 months. Each time you thaw you can freeze more to preserve stocks.
In principle Madhurima is right. However, the number of passages - not only for HEK293T cells - frequently depends on specifics of the laboratory and/or the person propagting the cells. We propagate HEK293T cells twice a week at split ratio of 1:3 and use bdbiosciences cell culture material. Cultures should be confluent after 3 days. Irregular cell layer indicated that the culture is not in a good condition. If this happens we thaw N2-stored cells.
As above - in theory they can keep forever, however, the longer they are in continuous culture, the more chance you have of contamination and mutation. Best practice is to cryopreserve your own stock form your earliest passages post thawing then return to these every 2-3 months. Each time you thaw you can freeze more to preserve stocks.
Hi Robert,
yes I agree with the above answers. In addition, for both HEK and HeLa cells I find that after about 30-40 (or 2 months worth) of passaging, the cells begin to change morphology and take on an overall elongated appearance. Sometimes a heavy passage of cells of this kind can help them recover but I have found that using these cells for experiments is usually not a good idea, as their responses to treatment and transfection seem to be altered. I hope this helps.
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