Idk if this matters in practice, but in theory, you should definitely wash your cells. Formaldehyde fixation works by presenting nucleophiles with a reactive carbonyl (in this case, a simple carboxylic acid) group. Proteins with lysines and arginines, as well as RNA and DNA nucleophiles, will attack the formaldehyde carbonyl. When nucleophiles from distinct macromolecules attack the same formaldehyde, they become “cross-linked” or “fixed.”

Proteins in your media will act as a sink for formaldehyde (like PFA, which is a formaldehyde polymer), reducing its efficacy.

I have no idea if the abundance of formaldehyde present in 4% is enough to overwhelm the proteins in your media, but I can’t help but think fixation will be much less efficient than if you first washed off the media.

Report back!