Dear Indra,

Cant be sure of apoptotic bodies till you run a caspase series. they look like atrophied. Use trypan blue and methylelene blue for death or living. Hope this helps.

Best

Mannan

Thank you sir for the opinion, I shall observe the cells closely first , before running some tests

I spent a lot of time adjusting my primary KCs cultures and my experince tells me that you shouldn't let your KCs to be that confluent. Keratinocytes stop cell cycle upon cell-cell contact and go into apoptosis ....if you keep cells in confluent state long enough all of them eventually will do it. If you want your primary KCs to proliferate nicely and stay in devission for longer you need to make sure that they don't cover more than 70% of the flask surface. None of this is collagen's fault. You only need coating for better recovery of cells when you put them from in vivo to in vitro. But once they start growing in vitro they don't need additionally coated flasks anymore ...just some proper ones for cultivation of adherent cells. 

Indeed Izabela, it reached 90% confluency yesterday..so I split them 1:3...i ll keep in mind to replate them at 70%, will be back in case those condition on the pics happen again

Hello,

Looks to me like there are also un-activated hepatic stellate cells here which are holding some lipid. I might be wrong, but if there are stellate cells, they will attach well to plastic.

You can split them much more than 1:3 ...i normally plate KCs 1000 - 2000 cells /cm^2. You give them optimal growth conditions if you split them in the consistent way taking care so they are never confluent. 

Thank you Izabela on normal condition I opt for 10-20 thousand/sqcm. However because of these condition on the pic..i am being cautious yesterday plating was approx. at 25000 per sqcm.

  I also feel that primary KCs requires somewhat a higher density to ploriferate properly, particlularly when they were not sufficiently acquired mutation, what do you think?

anyhow thank you for your advice and opinion

I think in case of keratinocytes particularly important is what medium you have for your cultures. To delay differentiation (and growth arrest) best is serum-free, low calcium. Success of the assay I do directly depends on the high proliferation rate of KCs. For the expansion phase I always plate cells in very low density. This way they expand really a lot and stay dividing for a while. Of course if you plate them in lower density it takes longer for them to fill up the flask ... but they will continue proliferating for longer if you don't "crowd" them. Cell-cell contact is a growth arrest/differentiation signal for KCs.

So for very low density culture how long will it take to reach a 70% confluency in your experience..I am assumming its not a weekly passaging...maybe 2-3 week ya?