Where my Vero cells has gone? - Mysterious dissapearing during experiment (Nov/15/2005 )

Hello, all

I tried to do an experiment of the interaction of ly protein with the Vero cell.
On the morning I have seen very nice pre-confluent cells on the surface of the Lab Tek Nunc 8-well chamber. After I have add my protein, after fluorescent conjugate (all incubation were 1h at 4)C), all washing between with the
Gibco DPBS ( without Ca, without Mg). After that I have not seen any cell on the well surface, even in the control well , where I have add nothing but DPBS.

Say me please, what can push my cells to gone?

Maybe it is important not to aspirate the PBS or medium quite rapidly? I did it with the pipettman

Maybe I should not tap the inversed plate on the paper tissue to wipe , as i do normally with ELISA plate, maybe Vero cells do not like it?

Maybe it is extremely important to do ALL EVERY second of the manipulation with the cells coveed by DPBS at 4°C?

Any suggestion where error could be?

help , please, to desesperate girl

our cells are pretty strongly adherent, but for making chamber slides to do IF, if you want cells in the slide at the end of the protocol you have to 1st) coat the wells prior to culturing them with the cells (we use usually use fibronectin, but other things like collagen and such will work)
and 2nd) you have to fix the cells prior to any washing or treating
and 3rd) tapping them is bad juju unless you are trying to uplift

I hope you can get it to work; good luck

Thank you very much for your responce.

Vero cells are fibroblast-like adherent cells, they detached normally only by trypsin, scraping, or EDTA.

Next time surely, I will not tap. But what could be a patent to suck the liquid from the 400 ul well? I tried much to not to touch the well surface by pipette tip, but in such a way it is difficult to take off all the liquid.

Maybe the problem was really in the heat shock. Next time I should take care to do all manipulations on glass, and to cool well my PBS solution.

Any other suggestions?

our cells are pretty strongly adherent, but for making chamber slides to do IF, if you want cells in the slide at the end of the protocol you have to 1st) coat the wells prior to culturing them with the cells (we use usually use fibronectin, but other things like collagen and such will work)
and 2nd) you have to fix the cells prior to any washing or treating
and 3rd) tapping them is bad juju unless you are trying to uplift

I hope you can get it to work; good luck

um....for fibronectin, we use 1 mg/mL in PBS, add enough to cover the well, and leave it in the 37C incubator for 1-4 hrs. the fn is clumpy so you vortex the $@#*&$ out of it immediately prior to coating.

There was a thread on here about a month ago, regarding different coating methods...you may do a search and try to find it? there were some very good suggestions

yeah, we fix with paraformaldehyde. I can give you the protocol if you like. I think some people fix with meoh, whatever...there are different ways and I am not sure which would be the best for your application