Fluo 5n

Fluo-5N was incorporated into 3a-reconstituted proteoliposomes and control liposomes by first thawing frozen liposomes at a ratio of ~1:20 (v:v) into modified sucrose formation buffer^{45 (link)} 25 mM HEPES pH 7.4, 150 mM KCl, and 262 mM Sucrose with a final concentration of 25 uM Fluo-5N (Stock concentration 5 mM in DMSO, AAT Bioquest). Next, the liposome mixtures in Eppendorf tubes were placed in a foam flotation in an ice bath and sonicated with a Branson Digital Sonifier 450 for 30 seconds total (10% Amplitude, 10 second pulse with 59 second wait time). Excess (unincorporated) Fluo-5N was then removed using microspin G-50 columns (Cytiva). The sample was then diluted into solution containing 50mM HEPES, 300mM KCl, and 2mM EGTA, pH=7.4 and plated onto poly-D-lysine (Sigma-Aldrich, 1mg/ml) coated 96-well plates, and centrifuged (5,000 × g for 5 min) at room temperature. Ca²⁺ influx was measured upon addition of a 50 mM HEPES, 285 mM KCl, 10 mM CaCl_2, pH=7.4 solution. Images were acquired every 3 seconds for a total of 150 seconds. Fluorescence intensity of each liposome was normalized to its average fluorescence prior to Ca2+ addition.