Biotek synergy neo2 fluorescent plate reader

Bio beads were filtered out using screening columns (Fisher Scientific, Hampton, NH, USA) and the proteoliposomes were mixed with 100 μM fluorescent dye 9-Amino-6-chloro-2-methoxyacridine (ACMA). Aliquots were incubated for 5–15 min, then diluted 20-fold (1:20) with Na⁺ buffer (20 mM Na-HEPES, 150 mM NaCl, 1 mM EDTA, pH 7.4 w/HCl). Dilution of the proteoliposomes serves to create a K⁺ gradient where the concentration in the proteoliposomes is much greater than outside. 20 μL of the dilute proteoliposome sample was mixed with 20 μL of Na⁺ buffer in a 384-well assay plate. Then, 8 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a H⁺ ionophore, was added immediately before starting the assay. Baseline fluorescence with CCCP was measured with excitation at 410 nm and emission at 480 nm using a Biotek synergy NEO2 fluorescent plate reader (Biotek Instruments, Winooski, VT). Ethanol was added to initiate flux and fluorescence was measured for 15 min. As K⁺ flows out of the proteoliposome CCCP transports H⁺ in, which quenches ACMA fluorescence in a dose-dependent manner. In this way, K⁺ efflux is measured as a function of H⁺ transport. 10–20 nM Valinomycin, a K⁺ ionophore, was added to measure maximal K⁺ efflux as a positive control.