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Problem with preparing unicellular suspension from MCF7 cells


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#1 veragesheva

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Posted 10 February 2012 - 04:36 AM

Hi there,
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!

#2 bob1

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Posted 11 February 2012 - 10:01 AM

MCF7 tend to clump together quite strongly. For your experiments scraping will not be suitable, too many cells will be damaged by the shearing from scraping. Make sure your MCF7 are not over-confluent, they should be a single layer of cells, not forming little bumps with multiple layers of cells (use a microscope!). This will probably, dependent on seeding density, mean that the plate/flask will not look confluent as you would normally define it, but will have open areas between colonies of the cells.

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.

#3 veragesheva

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Posted 13 February 2012 - 09:22 AM

Thanks very much for the answer! I will try to redo lifting off with trypsin/EDTA with probably more active pipetting.

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?

#4 bob1

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Posted 13 February 2012 - 02:18 PM

Partly, they do tend to grow in clumps as well. Have a look at the ATCC pictures: http://www.atcc.org/...hments/1980.jpg




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