I am using a 3% glyoxal pH 4.5 solution to fix adherent cells. I was previously using 1% PFA and established the best way to quench the reactive aldehyde groups was with 1M Tris pH 8 for 1 minute incubation. Does pH 8 Tris reverse glyoxal fixation? After quenching the glyoxal fixed cells like this, they began to lift off the plate. How do you reverse the chemical crosslinked Schiff bases created by glyoxal?
Hi Leslie Spitalny Schiff bases generated by both PFA and glyoxal aldehydes can be reversed by Tris up to a point. Schiff base rearrangement products (via Amadori) are practically irreversible.
Glyoxal is a more potent X-linker and is probably X-linking cells together more extensively than binding them to cell adhesion proteins on your plate. If your cells are coming off the plate in rafts, then maybe try less confluent cultures.
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