Hi all,
A little background: I'm working with a bone marrow fibroblast, HS-27A line and using a CRISPR KO kit to deliver the Cas9 enzyme with gRNAs via lipofection.
These cells do not like to grow alone and seem to have some quorum sensitivity. When single cells, most cells do not grow, and the ones that do end up dying and only debris are observed in the well after 7-10 days.
At this point, I tried many things - conditioned medium, semi-solid medium (methylcellulose-based), seeding a low number in bigger dishes, etc. Nothing has worked.
Since this is a KO, I can't screen the cells using any reporter - otherwise, I could do FACS. And since they're not under selection, I can't sort in bulk.
Do y'all have any thoughts or ideas on alternative ways I could get a monoclonal population isolated and expanded?
I appreciate it!
Hi, You have not mentioned about the medium that you are using and whether you use SFM with nutrients or FBS/FCS, if yes, what percentage? Did you check for contamination such as mycoplasma? Do you use P/S?
You stated that you plated a fewer cells in a larger dish, actually you have to do the opposite, that is plate more number of cells in a smaller dish or 6 well plate in order for the cells to grow efficiently.
Hi Chandra, thanks for the response.
I am using RPMI supplemented with L-glutamine, P/S and 10% FBS.
Cells have been tested recently for mycoplasma and results were negative.
The reason for few cells in large groups is to get cells spaced enough to pick individual colonies as they grow, while allowing them to still exchange secreted signals.
Hi Fabio Henrique, Okay. How much transfection reagent/ratio of Cas9 enzyme with gRNAs via lipofection, and how long you are keeping the transfected reagents, all these can affect cell survival and transfection efficiency. If you keep them longer or if you use excess of these reagents, especially as you plate cells sparsely, these might be the reasons for the cell death.
If your option is not to plate more cells, I suggest that you reduce the reagents concentration less than what you have used, and or keep 6-12 hours of transfection period, in case if you keep it longer and see if the cells survive better.
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