I am using collagen IV to coat flasks for PC12 cells to adhere and grow in serum free supplemented media. After slow thawing, i can see some gel in the collagen IV solution when pipetting up. I am using 0.05M HCL to dilute it and follow the manufacture's protocol to coat. after incubation, i can see some blots of gel formed on the base of flask. After seeding cells, the next day only around 5-10% of total cells adhere. I failed to coat the plate and am thinking the problem would be the gel which might contains most of the collagen.
Hi Dear Pin
I did this protocol too and id experienced this problem too.
when you are resolving col iv in HCl ,let it to defreeze col iv completely in RT ,under laminar hood sterile situation and after adding HCL resuspend it 5-6 times to obtain a complete homogeneous solution.
In this way after 24 hr seeding you have got some dead cell but dont worry.change the media slowly and let the cells grow in 3-4 days after. again change the media and repeat again . they will grow very nice.
note: to avoid miss the cells change 3/4 of media in every 3-4 days.
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