Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

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Abstract
The transverse location normal to the bilayer surface of a series of n‐(9‐anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 ≥ 3 > 6 ≥ 7 > 9 > 12 > 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.