Phrodo dextran

Cortical neurons at 12 DIV were transfected with LAMP-1 GFP as above, and then loaded with 10 µg each of pH-sensitive pHrodo dextran (Thermo Fisher P10361) and pH-insensitive Alexa Fluor 647 dextran (Thermo Fisher D22914) overnight. The following morning, dextran containing medium was washed with PBS, and neurons incubated in fresh medium for 3 h. Following drug treatments, neurons were imaged by fluorescence microscopy. The ratio of 668/585 was measured and converted to pH using an intracellular pH calibration kit (Thermo Fisher Scientific, P35379) as described in (Johnson et al., 2016 (link)). Only LAMP1-GFP and dextran-positive endolysosomes were included in the analysis.
An alternative protocol was used for studies of endolysosome pH in U87MG cells. Cells were incubated with the ratiometric probe LysoSensor DND-160 (Invitrogen L7545; 1 µM) for 10 min, washed three times with PBS, and then analyzed with fluorescence microscopy at excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm (Hui et al., 2012 (link)). Endolysosomes were differentiated from Golgi by adding CellLight Golgi-RFP (Invitrogen C10593; 2 µl/10 k cells) and incubating cells overnight at 37°C.

The lysosomal PH was determined using a fluorescence-labeled dextran dye (pHrodo™ dextran, Thermo Fisher) following the method established previously [13 (link), 19 (link)]. This dye emits strong fluorescence signal in an acidic environment while exhibiting minimal fluorescence signal at neutral pH. Briefly, NCL patient NSCs (1–2 × 10⁴ cells/well) were seeded in black, clear-bottom, 96-well plates and treated with compounds for 3 days. The cells were washed with NSC culture medium and incubated with NSC cultural medium containing 1 μg/ml Hoechst 33,342 at 37 °C for 30 min. Then the cells were stained with 100 μl/well 20μg/ml pHrodo™ dextran dye at 37 °C for 2 h. After washed twice with Live Cell Imaging Solution (Thermo Fisher Scientific), the cells were imaged in the IN Cell Analyzer 2200 imaging system using Cy3 and DAPI filter sets.

pHluorin imaging was carried out in HEK293T cells cotransfected with lyso-NpHR3.0/lyso-ArchT and pCMV-lyso-pHluorin (Addgene, #70113). All experiments were performed 24 h after transfection. lyso-NpHR3.0/lyso-ArchT was stimulated at 594 nm, while pHluorin fluorescence was excited at 488 nm by laser.
pHrodo dextran was used to monitor lysosomal pH changes in lyso-ChR2-expressing cells (Fig 2g–2l). Cells were incubated with pHrodo dextran (10 μg/ml; Thermo Fisher Scientific) for 6 h at 37°C and then cultured in medium without pHrodo dextran for an additional 1 h. Bafilomycin A1 (1 μM, MCE) was used to inhibit V-ATPase (Fig 2c, 2d, 2i and 2j). Imaging was performed with a Plan-APOCHROMAT 100×/1.4 Oil DIC oil immersion objective on an LSM980 confocal microscope (Zeiss).