Complete Isolation of Fibroblasts from Abdominal Wall - (Jun/04/2007 )
I am trying to completely isolate fibroblasts from the abdominal wall for a cell proliferation assay and have been unsuccessful. I was thinking about using a column with antibodies to fibroblast specific cell surface markers to do so, but am unaware of what markers would be exclusive to these cells. Also, is this feasible? I am an undergraduate new to cell biology techniques, so I apologize if the questions are obvious. Any help is greatly appreciated. Thanks!
I am isolating skin fibroblasts from 2mm skin biopsies and simply chop up the tissue into ~500uM diameter pieces, leave them in a tissue dish to adhere, then add DMEM/FBS. After 1 - 3 weeks the fibroblasts begin to migrate out of the tissue and once they are ~80% confluent I remove the biopsy from the dish and passage the fibroblasts as I would with any established culture. Any contaminating keratinocytes or endothelial cells do not survive well under the culture conditions so after 2 - 3 passages it is a pure fibroblast culture.
Hope that helps
Thanks for the reply. The problem I have is that I am trying to count the number of fibroblasts as soon as I extract the tissue from the abdomen so passages won't solve it. I need to figure out a way that will allow me to isolate the fibroblasts as soon as they are extracted.
Right now I can see 2 options:
1.) Use anti-rat fibroblast monoclonal antibodies to seperate them out (Problem: Too expensive because these antibodies against rat fibroblasts are not readily available)
2.) Place all cells in a culture dish and use cell cycle inhibitors so that when fibroblasts attach to dish surface, I can just wash the other cells/ particles off. This way, the cells don't divide and the number of cells I am getting for my assay is preserved. Don't know how effective this will be though...