TMEM16F is responsible for Ca²⁺–activated lipid scrambling in murine RBCs. (A) Schematic of the fluorescence lipid scrambling assay in RBCs. (B-C) Ca²⁺ (red)–induced phosphatidylserine (PS) exposure (AnV, green) in the RBCs from TMEM16F wild-type (WT; B) and TMEM16F knockout (KO; C) mice. Ten micromolar ionomycin was used to trigger Ca²⁺ influx and subsequent lipid scrambling. White boxes show an enlarged view. (D) Time-dependent lipid scrambling reported by AnV in the TMEM16F WT and KO murine RBCs induced by ionomycin. Error bars represent standard error of the mean (SEM). WT (n = 16 cells) and KO (n = 15 cells) from at least 3 biological replicates. (E) Schematic of PCLSF assay to simultaneously monitor TMEM16F lipid scramblase and ion channels activities (see “Methods” for details). (F) Representative images of lipid scrambling activity in WT and TMEM16F-KO RBCs under PCLSF. PS exposure was detected by membrane binding of the fluorescent AnV conjugates. (G) Representative current traces in WT and TMEM16F-KO RBCs elicited by a voltage step protocol from –100 mV to +160 mV with 20 mV increment. The currents were recorded ∼2.5 minutes after the whole-cell patches were established. (H) Representative time course of AnV signal on WT and TMEM16F-KO RBCs. (I) Fluorescence intensity of AnV signal at 3 minutes. Two-sided Student t test, ∗∗∗P < .001 (n = 5). (J) I-V relationship of the currents recorded in (G). The currents were normalized to cell capacitance. The results are presented as mean ± SEM (n = 6). (K) Current density at +160 mV. The results are presented as mean ± SEM. Two-sided Student t test, ∗∗∗P < .001 (n = 6).