We work with OKG4 gingival Keratinocytes between Passage 13-18 and cultivating them on Collagen 4 in 75cm Flasks and implement them with 2x10*4 cells on 96 Well with 0,6cm growth area and 5x10*5 cells in the flask again. Both products are from Falcon and coated with same concentration of Collagen 4.
There are immense differences in growth and morphology while both products were treated in the same way. The flask show much better results, while the cells in the wells could only be saved after several washings and medium exchanges.
Our Collegues work with HeLa- cells in the same incubator.
We are open for Helps in each directions!
Hi!
1.Is it necessary for your experiment to use collagen? Keratinocytes grow very well in tissue culture plastic without the need for coating. I would try to grow the cells in parallel in the flask and 96 well plate without collagen, to determine if the grow at the same rate first.
2. Do you coat the plates yourself? It may be easier to use already-coated plates as it may be hard to have consistent coating in such a small well.
3.In terms of number of cells plated, a final 70% confluence for keratinocytes (it will depend on which ones you use) would be approx 7.5x10^6 in a T75, and 0.3x10^5 for a 96 well plate/well.
I personally wouldn't plate anything below 20-30% confluence on either, as they will struggle to grow at that low concentration. So nothing below approx 2.5x10^6 in a T75 and 0.1x10^5 in a 96 well.
So I think first try to plate something on an uncoated surface, at min 25% confluence. Then if you are happy with the growth in both containers, you can try to switching to collagen, using already coated plastic.
Good luck!
Hi :)
Making a control without Collagen in the Wells seems to be a good idea it first.
I coat them by myself with 30µl Collagen 4 (20µg/ml) and vaccum it before implementation.
In The Wells i plate the cells between 1,5-2,5 x10*4 and in microscopic view it seems pretty dense. In the Flask they grow with lower density too.
I will keep you updated :)
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