Intracellular ph calibration kit

To estimate the cytosolic pH, MCF-7 cells were washed with RPMI without phenol red and then incubated for 30 min at 37  °C in the same solution, containing 1:1000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate (Thermo Fisher Scientific). Next, the cells were washed with RPMI without phenol red, and 2 h incubation with and without LNCs (0.5 and 1 mg/ml) was performed under cell growth conditions, in a medium supplemented with 1 or 10% FCS. Subsequently, the cells were harvested by Accutase^® Cell Detachment Solution (Sigma-Aldrich, St Louis, MO), centrifuged (300×g for 10 min at 4 °C), washed and resuspended in PBS. A standard curve was established by incubating cells as described above, and after detachment from wells, for 5 min at 37 °C with a series of pH calibration buffers (pH 4.5, 5.5, 6.5, and 7.5) supplemented with 10 µM valinomycin and 10 µM nigericin (Intracellular pH Calibration Kit; Thermo Fisher Scientific). Fluorescence intensity was measured by LSR II Flow Cytometer (BD Biosciences, San Jose, CA), using a 505 nm Ar laser filter and detected with the FL1 530/30 nm bandpass filter. At least 5000 cells were recorded in duplicates.

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.