I am doing cell transfection with plasmid DNA. I found that the transfection efficiency was related to the cell status definitely. For example, I used Huh7 cells these days. When cells were in 10cm dish it grow well but when I seed them in 24-well (10000/well, 1ml/well), I found that cells grow not good. The confluence was 50-60% overnight(confluence 80-90% & cell counts around 600000/ml in 10cm dish).
I guess that maybe it was because the cells were passage so many times, such more than 25 times.
Do I need to thaw a new cell?
Yes it could be. Mentioning your cell line would have helped here. The stability of cells across passages depends a lot on the type of cell line you are using. Some are highly stable lines and some are not so much.
This is often directly proportional to their transfection efficiency as well. If you enter a search query online with the name of your cell line you might find suggestions on the stability of the cells.
A straightforward method to calculate the optimum passage number does not exist. You will have to review literature for that. However, passage 25 is not considered that high according to my experience.
HI,
This is what we do.Thaw new cells and try to use maximum 5-6 passage. Keep your back up stock in Liquid Nitrogen.
Good luck
Anand
The number of cell passages has no a significant impact on attaining cell confluence efficiently if the cells are maintained good. The cell density on the dish should matter.
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