Why is fluorescence-minus-one not the same as an isotype control?

Why

In a ten-color panel, fluorescence from other nine dyes can broaden the negative distribution in the channel used for a dim activation marker. The corresponding FMO reproduces this multicolor spread and helps place an objective gate. An isotype control may reveal some issues with Fc-receptor or reagent-associated staining, but its affinity, concentration and fluorochrome characteristics do not necessarily mimic the actual antibody.

It does not reconstruct the same spillover environment as an FMO unless appropriately designed, and neither control proves binding specificity on its own. Include antibody titration, Fc blocking where needed and a known marker-negative population if feasible. For a clear bimodal marker, a dedicated FMO may be less critical after validation, whereas dim or continuous markers often benefit from it.

Record the gate and assess whether it stays credible across donor and treatment differences.