Ratiometric Lysosomal pH Measurement

Live cell ratiometric lysosomal pH measurements were conducted using a modified method from Saric et al^{64 (link)}, further optimized for high content imaging and analysis. WT and GRN-KO i³Neurons were maintained on 96-well dishes. On day 10, neurons were loaded with 50 μg/mL pH-sensitive Oregon Green-488 dextran (Invitrogen, #D7171), and 50 μg/mL pH-insensitive/loading control Alexa Fluor-555 red dextran (Invitrogen, #D34679) for 4 hours, before washing three times with PBS then chased overnight with neuronal media after PBS washes the day before imaging. These dextrans accumulate in lysosomes, and high-content microscopy quantification of their fluorescence enables ratiometric calculations of pH within individual lysosomes. Physiological buffers of known pH (4–8) containing 10 μg/mL nigericin were placed on WT neurons to generate a calibration curve. Live cell spinning disk confocal microscopy was performed using a Opera Phenix HCS System (PerkinElmer); calibration and sample wells were imaged at 63×; counterstaining was done with NucBlue Live ReadyProbes Reagent (Invitrogen, #R37605) to count and segment nuclei. Lysosome pH was calculated as ratiometric measurement of lysosomes (488/555nm), with subsequent calculation of the pH of those compartments based on the corresponding calibration curve. All analysis was performed using PerkinElmer’s Harmony HCA Software (PerkinElmer). Statistical analyses for all imaging data were conducted using independent student’s t-test.