Harvesting cells off of collagen gels - (Apr/08/2010 )
Does anyone have a detailed protocol for harvesting cells from fibrillar collagen gels? I coat the dish with fibrillar collagen and then seed cells. I have been using various concentration combinations of typsin and collagenase to lift cells off of the gel to count them, but have not been successful so far. My collagenase is from Gibco and it is called type II. I appreciate any help I can get as I am stuck
Biozisti on Apr 8 2010, 07:51 AM said:
Did you manage to get them off? When I worked with stem cells, we used to coat the flasks with Matrigel and then for harvesting, we aspirate the medium, wash the cells with PBS, add 7.5ml of collagenase to T75 (I think it is 100ul/cm2), incubate at 37 for 5min. Aspirate and wash with PBSw/o Ca/Mg, Add trypsin 7.5ml(again same - 100ul/cm2), incubate 5min, stop trypsin with medium with FBS, and collect the cells the usual way. Perhaps it depends on the cells.