It is common practice after surface staining cells for flow cytometry analysis to wash the antibody out of solution before analyzing a sample. I have tried analysis with and without washing the antibody out and notice no significant difference by flow. So is there a reason people do this step that maybe I'm just not noticing?
Two reasons for antibody wash:
- To limit non-specific epitope binding. All ABs have specificity. Washing is usually trying to remove weaker binding to non-intended targets.
- For flow, excess AB that is not bound, if also pre-conjugated to a fluorophore, may allow for excitation of the fluorophore while going through flow even though the antibody isn't bound to the cell type of interest.
If you had a very specific AB, and very little excess, you may not notice a thing. I'm sure there are additional reasons; these are the two that immediately come to mind based on my own flow cytometry experiments.