Cell dilution cloning - Cells cloning (Apr/04/2011 )

Hi,

I regularly do dilution cloning of cells in 150mm plates and 10cm plates. I dilute the cell suspension and seed only 500 cells in 30 ml growth medium in 150mm plate and ~ 100 cells in 10cm plate. In 1 week or so I have several small colony-like clones growing which I then pick carefully and transfer into 24 well plate. I did this successfully many times. (The result would be - some of them are single clones, some are not single, some are high expression, some are low expression of the desired protein - as assayed by FACS / ELISA etc.)
This time, my transfected cells - the pool population grows well but my clone - setting, even after 1 week, not a single colony is growing!! I repeated 2 times but still not 1 clone grows. I see nothing - no cells at all.
1) I did spike in more FBS to help the cells - no result.
2) I did increase the cell number from 500 to 800 or even, 1000. still nothing. What could be the reason? I do know that some cells need to have contact - then only they multiply. (meaning seeding very low, the cells die). I do see dead cell like debris. The medium in the plates is clear. I use the same bottle of medium for other cells in T - flasks and they grow well.
What could be the reason? Today, I shall do single cell cloning in 96 well plates, but I was just trying to figure out the reason!

Thanks in advance for your replies.

You could try conditioned medium as a mimic for contact with other cells. To do this take a 70-90% confluent flask of the cell type you are trying to clone and add fresh medium. After 12-24 hours, before the medium is depeleted, remove this medium and filter it. Store at -20 or -80 deg C. Add it to your clones in the place of normal medium.

OK. I'll try this definitely.

Thank you.

bob1 on Mon Apr 4 23:49:10 2011 said: