8 replies to this topic

[pana]

i grow B16 melanoma cells, they grow very nice in flasks but after i plate them for experiment in 24 wells dish after they attached and i began with any treatment,i found that they start to peel(my work mostly done under microscope) and they have the phenotype of dead cells.i have no explanation for that, have you encountered with such phenomenon?

[Biouday]

It is not clear what you treated with. It always suggest to check the cytotoxity of chemical which you treating before doing the actually experiment. i think peeling was because of toxic effect of the treatment.

Edited by Biouday, 22 May 2011 - 04:18 AM.

[pana]

[quote name='Biouday' timestamp='1306066672' post='110517']
It is not clear what you treated with. It always suggest to check the cytotoxity of chemical which you treating before doing the actually experiment. i think peeling was because of toxic effect of the treatment.
[/qu
but the peeling occurred before treatment
even begun

[leelee]

How confluent are they? I've not worked with B16 cells very much, but I did find that they did not like being 100% confluent and started to lift if I allowed them to get too confluent. Maybe try plating fewer cells into your 24 well trays?

[pana]

[quote name='leelee' timestamp='1306077060' post='110529']
How confluent are they? I've not worked with B16 cells very much, but I did find that they did not like being 100% confluent and started to lift if I allowed them to get too confluent. Maybe try plating fewer cells into your 24 well trays?

i dont think this can be the reason because peeling cells stil looks round and my cells looks dead

[bob1]

How are you determining that they are dead - did you try trypan blue as a simple assay?

Did you recently change supplier of FCS/FBS or plates?

[pana]

most of my work is done under microscopy so i can see how the cells looks like and they look as if the were treated and went through apoptosis.
i did not change a thing in the lab and the cells grow fine in flasks, i see this phenomena just in wells (24/96)

[bob1]

If I were you, I would be determining if the cells are actually dying before trying anything too drastic. If they went through apoptosis, you wouldn't be able to see the cells any more, you would just have floating debris. You certainly wouldn't get a sheet of cells peeling off.

If it is only a problem in the plates, try a new batch and check your seeding densities, which are a common cause of cells not attaching properly.

[briguy7]

maybe they just don't like whatever is coating the plate and so they don't attach--so as soon as you move them around on the microscope to view them they start peeling off. Try a different type of plate