Acridine orange

OV202NTC and OV202Sh1 cells were seeded in 4-well tissue culture chambered slides and grown overnight. Cells were stained the next day with acridine orange (1 μg/ml; Molecular Probes, cat# A1301) in a serum free medium for 15 minutes at 37 °C. acridine orange was removed and images were captured in Zeiss LSM microscope. Red fluorescence indicates the acidic vesicles as described earlier68 (link).

Lysosomal V-ATPase activity was assessed with acridine orange staining. acridine orange uptake represents lysosomal V-ATPase-driven pumping of hydrogen ions into the lysosomes^{36 (link)}. After the indicated treatments, HEI-OC1 cells were washed with PBS and incubated with 1 μM acridine orange (ThermoFisher, A1301) in the cultured medium for 30 min at 33 °C. The acridine orange was then removed, and the cells were examined under a confocal microscope (LSM 880, Zeiss, Germany). The cytoplasm and nuclei of stained cells showed bright green fluorescence, whereas the acidic autophagic vacuoles showed bright red fluorescence^{10 (link),37 (link)}.

Fluorescent probes that stained different cellular compartments were used to characterize the origin of the vacuoles. The dapoxyl ER-tracker blue-white dye, the Lyso-tracker red dye, the Mito-tracker green dye, and 10 kDa and 70 kDa Texas-Red labeled dextran were all obtained from Molecular Probes (Life Technologies, Merelbeke, Belgium). We also used Lucifer yellow (Lucifer Yellow CH, lithium salt) from Biotium (Fremont, CA, USA) and acridine orange from Sigma-Aldrich. Briefly, the cell seeding and treatment procedures were similar to the ones used for the phase contrast microscopy (Section 4.3.1.). The dye solutions were simply added to the culture medium 1 h before the end of the treatment periods, excepted for the Lucifer yellow and both dextrans 10 kDa and 70 kDa, which were added for the whole duration of the treatment. The concentrations of the dyes were as follows: ER tracker, 0.5 µM; Lyso tracker, 75 nM; Mito tracker, 200 µM; Lucifer yellow, 100 µg/mL; acridine orange, 1 µg/mL; and dextrans 10 kDa and 70 kDa, 125 µg/mL; At the end of the treatment period, the procedure was similar to that of phase-contrast microscopy to take pictures of living cells with the Imager M2 fluorescence microscope (Carl Zeiss) coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss). The experiment was realized at least two times in duplicate.

The rbbp4 cDNA was cloned into the expression vector pT3TS. A 1 μg linearized vector was purified with the PureYield Plasmid Miniprep System (Promega, A1223) and used as template for in vitro synthesis of capped mRNA with the Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, AM1348). In vitro synthesized mRNA was purified with the RNA Clean and Concentrator Kit RCC (Zymo, R1013). Single‐cell zebrafish embryos were injected with 100 pg or 300 pg rbbp4 mRNA. At 24 hpf, the embryos were incubated for 5 hours with 100 μm Camptothecin (Sigma‐Aldrich, C9911) or DMSO as a control. Embryos were incubated in 10ug/ml acridine orange (Thermo Fisher Scientific, AC423340010) in embryo media for 30 minutes. acridine orange‐labeled living embryos were rinsed with embryo media and immediately imaged on a Zeiss Discovery.V12 stereomicroscope using a Cannon Rebel digital camera and EOS Utility software.

The acidification activity was measured with Acridine orange staining assay according to previously described methods40 (link). Osteoclasts were incubated in α-MEM with 10% FBS containing 5 μg/mL Acridine orange (Dojindo) for 15 min at 37 °C, then washed three times with PBS. The cells were incubated in fresh medium without Acridine orange for 10 min at 37 °C and observed by confocal microscope (LSM 710 [Zeiss]) with an appropriate filter set (excitation: 450- to 490-nm band-pass filter and emission: 515- to 565-nm band-pass filter or 600-nm long-pass filter). Images were analysed by using MetaMorph software.