We are carrying out ICC staining on iPSC derived microglia on 96 well imaging plates.

During the staining procedure we seem to be detaching a lot of the cells no matter how careful we are during the washing steps etc.

At the moment our cells grow on PDL coated plates and fixed for around 20 minutes in 4% PFA.

We have reduced the number washing steps to try prevent this as well trying a Microplate washer from the recommendation of others.

We still seem to be losing a lot of cells from the center of our wells, any suggestions on how to prevent this?