Hi all,
I am doing a transient transfection to my cells with GFP-tagged plasmid and sending them to be FACS sorted and I need to replate them once I get them back. This is my first time ever doing this kind of experiments. would you please kindly help on how I could replate them? is it casual cell culturing? I heard that you send your plate and get a tube back?! would you be so kind to elaborate? Thank you
1. Collect your sorted cells in 100% FBS (to maintain high cell viability).
2. Spin down your sorted cells and resuspend preferably in DMEM/F12 1:1 mix supplemented with 20% FBS. Replate your cells as usual.
Good luck
I do the same as Rahul (above), but ensure that the FBS is temperature controlled as well as the sample in the FACS. The FACS should have a setting, mine are maintained at 5C.
I spin immediately after sorting, but using a lower RPM than usual as FACS can be stressful for the cells. I usually spin at 1200RPM, and reduce to about 750RPM. this may not make a huge difference but I like to minimise cellular stress. if you do reduce the RPM, be careful when removing the supernatant as the pellet may not be as tough as if you has spun at a higher RPM.
Depending on the expression of your plasmid, your cell count after sorting can be very low. for example, i routinely sort roughly 15x10^6 cells and usually yield about 4x10^4 cells. seed your cells appropriately, and be aware that if you have a low cell yield they'll take a long time to expand depending on their normal growth rate.
Good luck!
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