Primary cultures of breast carcinoma for FACS - (Jan/16/2007 )

Hi all,

I wondered if anyone has any experience of trying to culture breast tumour cells from lumps of biopsy. The problems I envisage are:

1. How to disagregate/disassociate the tissue into single cells - is enzymatic digestion of contaminating stromal cells reasonable quick and effective?

2. Are the tumour cells easy to gate on the Forward/side scatter (going to also use cytokeratin positivity as a defining feature)

and a more general problem is how to harvest the cells, which presumably will adhere, from the 96 well plates I'm culturing in. I only want to culture the cells for 24 hours so would prefer a reasonable quick method of setting the cultures up.

Any advice, experiences, nightmares would be greatly appreciated!

Thanks,
Giles

Here is a link to a review of the HMEC system. Hope it helps!

http://www.lbl.gov/LBL-Programs/mrgs/review.html