How to seed cell in plates - (Apr/08/2007 )

In the beginning, I also endedup with hard clumps of HEK293 after centrifugation. Here is how I solved this problem.

1. Split when cells reach to a max of 95% efficiency.
2. Remove media and wash cells 1X with PBS at RT
3. Add 2ml trpsin-EDTA (cold, right from refrigirator) on a T75 flask. Wait for 30 sec and remove all trypsin
4. Put flask at 37oC for 3 min.
5. Tap the flask to dislodge the cells, add 10 ml media (DMEM/10% FBS).
6. Collect cells in a centrifuge tube (placed on ice previously) and centrifuge at 4oC
7. Add 10ml fresh media, mix using pipette 5-6 times and put the tube on ice (at this point I count my cells using coulter countr).

Either it was gentler treatment with residual Trypsin-EDTA or keeping cells on ice, cells never clumped together. If the cells are kept on ice for more than 30 minutes, they do kind of clump but can be resuspended just by swirling the tube.

As you know, HEK293 loosely adhere to the surface, however, if you find your cells unusually sticky, dump them and start with low passage number cells from the stock.