Hi, I am attempting to culture 3T3-F442A cells and carry out IF staining.

I'm testing a variety of different compounds on the cells (e.g. lopinavir, clozapine, olanzapine etc.) Up until recently, I have had no problems with cell adherence. From the past three attempts, after the first drug addition, the cell monolayer detaches and clumps together into one large mass of dead cells.

I don't think this is attributed to the drug having a cytotoxic effect. We have a control well of media only and still see the monolayer detaching.

I also don't think this is attributed to over-confluency, as the cells are ~80% confluent when it comes to adding the drug.

I autoclave and also sterilise the coverslips in 100% ethanol before adding to the plate. I use a 1:30 dilution of rat tail collagen in de-ionised water to coat each well/coverslip. I then leave the plate in the incubator for a minimum of 1 hour.

Any suggestions or ideas to resolve this issue would be greatly appreciated!