collagen II flask coating: how? - (Sep/01/2005 )
hello,
I want to know if some one had ever coated a colture flasks with collagen before seeding cells and whta is the correct procedure to ensure the collagen
adhesion and to avoid him to be washed out.
TY
I routinely use Type I collagen to coat culture dishes for adhering my cell line of interest (which normally grows in suspecsion) for long-term experiments. Type I collagen is soluble in 0.1N acetic acid, and I suspend it to a concentration of 1mg/mL. You'll need to know the molarity of the stock acetic acid to figure this out. The solubilized collagen is then aliquoted and stored at -20 until needed. I understand it can also be stored at 4 degrees.
In order to coat dishes, the collagen is diluted with 30% EtOH at the desired concentration. I'd run a quick O/N experiment to identify the lowest concentration of collagen that still promotes good adherance for your cell line. For me, ~4ug/cm2 works well. I add about 1.5mL of EtOH diluted collagen solution to each ~60cm2 culture dish to be used for the experiment. The dishes are then manually rocked to coat the entire bottom and left to dry for several hours to O/N unlidded under the culture hood. I typically use my plates immediately after drying, but they can be stored at 37 degrees for a day or two at most.
Cells can then be removed from the plate by either forced pipetting or scraping.
Knock yourself out... I am pretty tired of coating dishes myself...
In order to coat dishes, the collagen is diluted with 30% EtOH at the desired concentration. I'd run a quick O/N experiment to identify the lowest concentration of collagen that still promotes good adherance for your cell line. For me, ~4ug/cm2 works well. I add about 1.5mL of EtOH diluted collagen solution to each ~60cm2 culture dish to be used for the experiment. The dishes are then manually rocked to coat the entire bottom and left to dry for several hours to O/N unlidded under the culture hood. I typically use my plates immediately after drying, but they can be stored at 37 degrees for a day or two at most.
Cells can then be removed from the plate by either forced pipetting or scraping.
Knock yourself out... I am pretty tired of coating dishes myself...
hi,
I grew the PC12 on collagen coated flask. During subculturing, I have difficulty getting majority cells detaching with trypsin up to 4mins.
In the end I have to use scraping.
Up to how long maximum can we expose the cells with trypsin?
Does scraping (gently) recommended?
You can give PBS wash to your collagen coated plates just before seeding cells. If trypsin is not working you might want to use collagenase to detach the cells (as plates are coated with collagen).
hi--
I have gone with the collagenase method to harvest the cells that had been grown in collagen-coated wells. In about 20 min I obtain the cells with a 2mg/ml collagenase solution (and a little help scraping gently with pipette tip). My question is whether I get rid of all the collagen protein when centrifuging the cells...I need to determine total cell protein to normalize my data...Anyone can give their words of wisdom?