Dear Pieter,

I would suggest to spin down the cells, remove the supernatant and resuspend the cells in a small amount of ECL-gel (20-50uL). Than you can transfer it on a coverslip and let it polimerize. Try to cover the whole surface instead of having a thick dot of ECL on your CS (easiest on ice...). 

It might be that the incubation and washing times have to be increased due to the lower movement within the gel.

But you could be sure that you don't loose the cells.

Best

Lukas

Hello Pieter

For non-adherent cells in sparse quantities, I usually spin them down, resuspend in about 100uL complete medium and seed them on coverslips pre-coated with collagen/Poly-L-Lysine (this will depend on the type of cells you are using. eg: RBC stick quite well on Poly-D Lysine coated coverslips). I then proceed with fixation and ensure all washing steps are extremely gentle (which means no vigorous shaking or pipetting). 

Another method, that I use with Plasmodium infected RBCs (non-adherent, few and super fragile) is staining in solution and use centrifugation steps to wash. Mounting is performed in the end on Poly-D-Lysine coated coverslips. Though one needs to optimize the centrifugation speeds to ensure minimal loss of sample, as well as greater quantities of antibodies are required. Personally I would recommend the former method.

Hope this helps. Good luck.

Anyone have protocol for Erythrocyte and other blood cells confocal microscopy slide preparation ?