Problem with preparing unicellular suspension from MCF7 cells
Posted 10 February 2012 - 04:36 AM
I have a big problem with preparing a unicellular suspension from MCF7 breast cancer cell line. I need to do this because I need to establish a SCID model of human breast cancer injecting the mice with exact number of cells.
The problem is that after I take off the cells from the flask (I have tried different things: Trypsin, Acutase and also a scrap tool), centrifuge and resuspend them I can't count them because cells form floating islands and groups, which doesn't allow correct counting. The same is happening when I am using C-26 colon cancer cell line.
If anyone knows how to solve this problem please do help me-this stops my work from the very begining!
Posted 11 February 2012 - 10:01 AM
Trypsin/EDTA will be your best bet. For this to work you need to make sure that you are not over-trypsinising the cells - just wash the cells once in PBS, then add 1-2 ml of trypsin per T-75 and incubate only until the cells lift off - do NOT do this for a defined time, as this usually causes problems in my experience. Tapping the side of the flask will bump cells off that are weakly attached. You will then need to pipette the cell suspension up and down a few times to form the single cell suspension.
Posted 13 February 2012 - 09:22 AM
But what I've noticed: my cells are not growing normally in monolayer but they tend to form bumps and groups. I use MEM media, 10% FCS.
Could this be due to the fact that I failed to make them a unicellular suspension before freezing and probably freeze them as groups of cells?