3-((3-((9-(Diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)propyl)(dodecyl)(methyl)ammonio)propane-1-sulfonate (NR12S, Cat No 7509, Tocris) was used to measure membrane fluidity. For live imaging, parasites were incubated 60 min with or without treatments in a Panasonic tri-gas incubator set to 5% Co2 and 2% O2. Before imaging, parasites were incubated 30 min with 0.05 μM NR12S after exposure to different treatments, washed out with 1XHBS. Images were acquired on a DeltaVision Elite microscope using a 60X, 1.4-numerical aperture objective with a PCO Edge sCMOS camera, YFP and mCherry filter set, and images were deconvolved using SoftWorx (API, Issaquah, WA).
For plate reader assay, cells were resuspended in cold 1X HBS (HEPES-buffered saline) and dilutions were made after measuring cell density using a MOXI Z mini Automated Cell Counter Kit (Orflo, Kenchum, ID). To measure NR12S fluorescence intensity, 20,000 cells were loaded into black polystyrene, clear bottom 96-well plates (Greiner). The fluorescence intensity of NR12S were measured using TECAN SPARK plate reader with excitation wavelength 520 nm, and emission wavelength 560/630 nm.
For plate reader assay, cells were resuspended in cold 1X HBS (HEPES-buffered saline) and dilutions were made after measuring cell density using a MOXI Z mini Automated Cell Counter Kit (Orflo, Kenchum, ID). To measure NR12S fluorescence intensity, 20,000 cells were loaded into black polystyrene, clear bottom 96-well plates (Greiner). The fluorescence intensity of NR12S were measured using TECAN SPARK plate reader with excitation wavelength 520 nm, and emission wavelength 560/630 nm.