My problem is that the naive CD4+ cells do not want to proliferate after electroporation.
I leave them in the strip for 30 minutes and after transferring the cells into a 96-well plate for another 3 hours.
First after these 3,5 hours-long resting, i add my cytokine mix to the cells.
The non-electroporated controls proliferate after 3 days really nice, but the electroporated ones don´t. Does anybody have any ideas why the electroporated naive CD4 T cells do not proliferate and how can I increase the recovery of these cells?
Thank you for any ideas in advance.
Hi,
do you have control electroporated cells (equal amount of cells in the same suspension medium with Cas9 without sgRNA and sgRNA without Cas9)?
This should tell where the problem is. Is it in the components used or the electroporation itself.
Besides the no-proliferation problem, are your cells alive after electrophoresis? You could try Trypan blue staining or an equivalent test and check. If not alive you should do the electroporation with milder conditions
Dear Mateo,
Thank you for your feedback.
Yes, I have controls without sgRNA, but these cells seem to proliferate even less compared to the condition where I have sgRNA+Cas9 together.
After electroporation I have to induce their differentiation into iTregs, therefore I also have controls which are not electroporated at all and these cells proliferate without any problems. After the differentiation I perform flow cytometry analysis and cells seem to have a good survival. The problem is, I have so few cells and they don´t proliferate.
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