Cell count on MCF-7 - (Jul/06/2005 )
I am doing a cytotoxic assay on MCF-7 cell line.
I have a problem with clumping cell after trysinization, so I couldn't count it properly. Any suggestions, please.
Wash it with PBS several times, then add trypsin, incubate at 37 C for a couple of minutes, then take out and watch under microscope. Once it is detached from the plate, gently pipette up and down. Then add medium then continue with counting.
You might also want to try switching to Trypsin/EDTA
Hey....after trpsinizing (in batches if you have lotsa wells to count) resuspend them in serum free media or pbs. I generaly do my countings in 24 well plates and add 100 ul of the trypsin wait for 5 mins and add 900 ul of SF media or PBS. Idea is to not llet the cells sit for a long time.
In my experiences with MCF-7, they clump worse if you tap the dish to release them when they are trypsinizing. Also, if your culture is really confluent at trypsinization, there will probably be nothing you can do. I would always try to use them before they got packed pretty tight. That way you can avoid some of the clumpy-ness.
When I'm using MCF7 cells, I try to get them into single cells by pipetting with the serological pipette abt 50 times - not harsly, but just up and down. And when I look down the microscope, 80-90% are single cells. Also, if you do it right, you can get good viability too - initially i used to get lots of floating cells, but have sinced improved with practice.