Hi, I'm currently transfecting my cells using 4D Amaxa electroporation and the cells recover beautifully post-transfection. However no matter how many times I try or whatever method I use the cells always die in the 48 or 96 well plates after colony picking. I've tried using antibiotics to do it outside but mostly I do the colony picking inside the culture hood so it's unlikely to be contaminated. I've tried using colony picking tips and the smaller 10ul pipette tips and I've also tried using accutase and without accutase but all the cells die.
I've also tried washing the cells with DPBS and drying the well and used only 10ul of DPBS around each colony before scraping them and pipetting the liquid back in together with the fragmented cells (Only this method gives a growing cell but really slow probably due to antibiotics, and it requires quick picking within 10 minutes or so before the DPBS crystallises hence not really optimal).
I've also tried manually circling the colony with a marker and simply using a pipette to scrape the well within each circle and quickly pipetting out 10ul into a well in a 96 well plate while minimizing pipetting. None of them works for some reason, it's really strange. I use Laminin 521 which works well for culturing cells, I've tried 48 wells plate, I've tried filling all the wells in the 96 well plate to prevent evaporation, I've tried using 50:50 media half is fresh the other half is from old media and none of them works.I've also tried adding Y2 and still the cells don't survive. The transfected cells are grown in a 6 well plate prior to colony picking.
Any idea/suggestion would be really helpful. Thanks in advance! :)
Hi Dee, I assume that these are not transient transfections but stable transfections with a selection marker, no?
And the cells are alive and kicking until you try to isolate the clones. But the cells might still be trying to adapt to the inserted gene that might be throwing their metabolism into whack.
My advice is to leave the cells alone. Let them adapt, become confluent and then do clonal selection.
Hi Steingrimur,
Yes, these are stable transfections with Cas9 RNP and donor templates. The cells recover beautifully with normal growth rate after transfection and reach 90% confluence after about 5 days so I doubt it's the cells. It's something about smaller plates that is causing the cells to die and I can't figure out just what it is
Hi Dee, check the specs on the smaller plates you are using. Plastic plates can be very different.
Normal plastic plates for TC allow tight for tight binding of the adhesion protein vitronectin, which has an RGD sequence that engages av integrins and is a survival signal.
Bacterial plates do not support adhesion proteins from serum and cells don't attach and die.
If you have 48-well TC plates, incubate them with 10% FBS for ~30 min before transferring the cells.
96 well plates can have the same problem. If they do not support vitronectin binding, the cells wont adhere and die.
Hi Steingrimur,
Thanks for the suggestions! But the plates I use is the same brand (Corning) as the 6-well plates I use to grow the cells and they are meant for tissue culture. Only when transferred to smaller wells do cells have a problem to attach and grow. I use Laminin 521 for cell attachment in all the plates I use. I doubt that it's the specs for the plate as no one ever complained about the plates before but I have no idea what is it about the smaller diameter that is disrupting cell growth. I'm checking the 96 well plate by growing normal cells (no transfection) to see if they grow and to see whether there's a problem with the regulation of the incubator. Do let me know if you have other suggestions!
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