Lysosensor dnd 160

The cells were inoculated on a 96-well plate. After growing to a suitable density, preheated (37 °C) medium containing 1 µM LysoSensorDND-160^® (Thermo Fisher L7545) was added. The cells were incubated for 5 min at 37 °C, and then the medium was removed and replaced after washing the cells three times with PBS. WellScan mode was used to read the plate in BioTek Citation 5 (BioTek, Winooski, VT, USA). Fluorescence was measured using Ex360nm/Em450nm and Ex380nm/Em540nm excitation/emission wavelengths, and the lysosome pH was determined from the ratio of Em540 to Em450.

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.

Cells were incubated with a medium containing the specified drugs for the indicated times, and then stained with 2 μM LysoSensor DND-160 and 100 nM LysoTracker Red DND-99 (Invitrogen) for 10~30 min. After washed with probe-free medium, the samples were viewed using fluorescence microscopy (Zeiss, Axio Vert. A1).