Endosome and lysosome pH

Mouse hippocampal neuronal cultures were analyzed at DIV 8. For each animal, 3 or 4 wells were seeded in CellCarrier-96 Ultra microplates (PerkinElmer) for replication purposes. Lysosome pH was measured using a protocol adapted from Johnson et al. (2016). At DIV 7, cells were incubated with 0.1 mg/ml each of OG 488-dextran (OG-dextran) and TMR-dextran (TMR-dextran) for 2 h at 37°C. They were washed 3 times with 1× PBS and then chased overnight in supplemented Neurobasal media. Before live imaging, cells were incubated with Hoechst-33342 (1:1600) in supplemented Neurobasal media-minus phenol red for 10 min to label cell nuclei. They were then washed once with 1× PBS and imaged with supplemented Neurobasal media-minus phenol red. The fluorescence ratio was converted to absolute pH using a pH calibration curve. The calibration curve was generated by imaging pH standards (e.g., 3.5, 4.5, and 5.5) in a calibration solution (125 mm KCl, 25 mm NaCl, 10 µm monensin, 25 mm MES, and adjusted to a final pH using 1N NaOH or 1N HCl). For bafilomycin A1 experiments, 100 nm was added with Hoechst-33342 in supplemented Neurobasal media-minus phenol red for 10 min. Cells were then imaged in supplemented Neurobasal media-minus phenol red with 100 nm of bafilomycin A1. Endosome pH was measured in primary hippocampal neurons at DIV 5 as previously described (Ouyang et al., 2019).