Fluorescence-Gated Flow Cytometry Approach for Measuring Lipid Flippase Activity in Mammalian Cells

P4‑ATPase lipid flippases establish transbilayer lipid asymmetry in eukaryotic membranes, with increasing evidence indicating a role for interacting partners in regulating flippase activity. However, assessing interactor‑dependent effects on flippase activity in mammalian cells is complicated by heterogeneous protein expression and background lipid transport. Here, we introduce an expression‑based gating strategy for in‑cell NBD‑lipid uptake assays that improves sensitivity and interpretability under transient expression conditions. Using this approach, we examined the influence of the putative interactors VAMP3 and TMEM230 on the ability of ATP11C to transport phosphatidylserine (PS). VAMP3 exhibited no detectable effect on NBD‑PS uptake, whereas TMEM230 produced a strong inhibitory phenotype. The expression‑based strategy increased resolution between ATP11C variants and substantially reduced the number of events required for reliable analysis. Together, these advances provide a framework for systematic, cell‑based investigation of regulatory interactions governing mammalian P4‑ATPase function.