I would recommend saving any supernatants and centrifuging them to keep any detached cells. Then simply remove the supernatant from the tube and resuspend it with the next buffer in the procedure and return to the original well. Do that for steps 1 and 4.

Dear Nina

I agree with @Mateo that you must avoid losing detached cells within the supernatant. I suggest to perform the live/dead cells assay before detachment of dying cells. Besides, you can probe the existence of suspended cells in each step by the bright field microscopy. It is worth noting that you can also use flowcytometry-based analysis to estimate the percentage of dead cells utilizing PI staining. For this purpose, you must avoid discarding supernatant (i.e. the media containing suspended cells should be kept and no washing step should be done). Good luck

Hi Nina,

I agree with Alireza and Mateo. I can also give Hoechst 33342 in combination with EthD-III a try. Hoechst 33342 is membrane permeable and would stain all cells (nucelli) and while EthD-III will only stain the cells with a defective membrane. Both dyes could stay in the culture medium, so that there would not be a problem with washing. I have used them in a live cell imaging experiments to document different kinetics of dying cells.

Best wishes

Sönke