2 replies to this topic

[chicken]

hi

i want to know, if there any one working with huh-7 cell line or have any experience,so please guide me.

we are growing huh7 cells in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) , 100 units/ml penicillin/ streptomycin,we do not add   2 mM L-glutamine and haven't treated with any anti mycoplama decontaminat.

our cells after trypsanization grow so good, for transfection when we made 6-well or any other plate, cells suddenly dislodge after 1X pbs wash, even dislodge if wash with dmem media. we wash soooooooooo carefully , no pippette pressure .

but cells detach as full layers   

we still cant sort out, what would be the possible reason.

one thing that is strange  our cells background is not clear ,it contain very blakish granules type small structures,even cell looks like full of granules.
is it mycoplasma

can any one guide me.and help me in sortin out

[piwi]

I've the same problem with my Cos7 cells. After trypsinization they grow quite well but in a 6-well plate they detach as full layers when they're washed even as gently as possible (exactly like your cells as you describe), so I can't perform immunofluorescence assay because finally I have no cell!!! I've check the cell at the microscope after each time I wash them, and actually the cell number decrease dramatically.
I've no solution at the moment. Can anybody help?
Thanks a lot

[SciCell]

piwi, on 28 October 2010 - 05:32 AM, said:

I've the same problem with my Cos7 cells. After trypsinization they grow quite well but in a 6-well plate they detach as full layers when they're washed even as gently as possible (exactly like your cells as you describe), so I can't perform immunofluorescence assay because finally I have no cell!!! I've check the cell at the microscope after each time I wash them, and actually the cell number decrease dramatically.
I've no solution at the moment. Can anybody help?
Thanks a lot

I have similar problem with my 293-Eco cells! The cells easily detach. But all the detached cells are live. They are not dead. But like you said we cannot use the cells for specific experiments. But in general the low adhesion cells peel off and detach if they are fully confluent. Maybe you should try splitting at a low density. I did that and saw some improvement. For example even if the protocol says, seed 7e5 cells in one well in 6 well plate, I seed only 5e5 - this might work but again, we need to modify the protocols.

Thanks,