I am busy generating a KO cell line with Jurkat suspension cells.
After transfection I perform single cell dilution (limiting dilution) to approx. 70-100 cells per 96-well plate.
It seems that none of the cells have survived this dilution.
Any pointers ow I can up the survival?
P.S. I have already performed control electroporation experiments and survival rate is very high after transfection. Transfection pools grow out very well.
But the cells die off once I perform single cell dilution.
How can I get a pure colony of Jurkat cells
Hi Nikki,
basically limited dilution in a 96-well plate is the easiest way to go if you want to obtain a clone from a single Jurkat cell. Did you see cells in your wells after seeding - this would be the first troubleshooting step I could think of? If there are cells, you should optimize growth conditions (increasing serum concentration?). If there are no cells from the very beginning, something might be wrong with your dillution procedure.
Just a thought - I have never tried that myself but maybe you could also resuspend your cells in soft agar (~0,3%) and see if it is possible to get colonies this way (similar to an anchorage independence growth assay)? Isolating colonies and expanding them further can be done after isolating the clone by "microplugging" with a capillary...?
Best,
Christian
Robert Adolf Brinzer
I washed the cells with 1x PBS.
Resuspended the cells fresh RPMI medium (10% FBS, 1% P/S, 1% glu)
First of all, this is a common problem and you are not the only one having difficulties to obtain single cell clones from Jurkat.
You might try to use conditioned medium.
And here you find a previous discussion regarding this topic with additional tips & tricks.
https://www.researchgate.net/post/How_can_I_expand_a_transduced_single_jurkat_cell
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