How to harvest highly adherent target cells after a co-culture killing assay for Flow Cytometry?
Hi everyone, I am setting up an overnight co-culture cytotoxicity assay using strongly adherent target cells (solid tumor models) and suspension effector cells. My final readout is flow cytometry (FACS), using a cell tracker dye to gate the target population and a viability dye (like 7-AAD/PI) to measure specific cell death. The issue: After the overnight incubation, the target cells are tightly attached to the bottom of the flat-bottom plate. Standard mechanical detachment (pipetting) is not effective enough—cells stay stuck, and I risk physically destroying the fragile, dying target cells. On the other hand, I want to avoid standard strong trypsinization at the end of the assay because I am afraid it will completely lyse the compromised target cells or interfere with the viability readout. Has anyone optimized a gentle detachment protocol for very sticky cells in a similar flow cytometry setup? Do you use alternative, gentler dissociation buffers (like Accutase or specific non-enzymatic buffers)? Does a cold shock help? Or is it generally better to just perform the entire overnight co-culture in round-bottom (U-bottom) / ultra-low attachment plates to avoid the attachment issue entirely? Thanks in advance for any tips or methodological advice!