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11 protocols using acridine orange

1

Fluorescent Staining of Apoptotic Cells

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Cells from different co-cultures on coverslips were stained with Acridine Orange AO (Roche Diagnostics GmbH, Mannheim, Germany) working solution (0.01%) at low pH, contrasted by crystal violet and were used for examination under fluorescence microscopy (excitation/emission: at 520–650 nm). Briefly, the cells were washed with PBS and stained with AO for 5 min at room temperature. Then, after washing with PBS, contrast staining with crystal violet was performed for 3 min in the dark, and was followed by three times washing with PBS. Acridine Orange (AO) was used to identify engulfed apoptotic cells and visualized under fluorescent microscopy (Axioskop 40 Zeiss, Oberkochen, Germany). Experiments were performed in triplicate.
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2

Embryonic Apoptosis Assay Using Acridine Orange

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Embryos were dechorionated and anesthetized in E3 media containing 0.02% Tricaine (Sigma-Aldrich, St. Louis, MO, USA). Embryonic apoptosis by chemical toxicity was measured using the vital dye, Acridine orange (Sigma-Aldrich) after exposure to chemicals. The embryos were stained with 3 µg/mL Acridine orange in E3 media at 25 °C for 30 min with shaking in the dark, washed 3 times with E3 media, mounted in 2% methylcellulose and observed alive using a Zeiss LSM780 NLO confocal microscope (488 nm Ar-laser excitation and 500–560 nm emission filter, Carl Zeiss, Overkochen, Germany).
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3

Visualizing Lysosomal Structures in Corneal Fibroblasts

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It has been established that acridine orange accumulates in acidic organelles.20 Cells were cultured on a cover glass slide chamber, followed by the designated treatments. Briefly, corneal fibroblasts were exposed to 0.5 μg/mL acridine orange (Sigma‐Aldrich) for 15 minutes at 37°C.21 After washing with PBS three times to remove excess acridine orange, the lysosomal structures were visualized with a Zeiss LSM700 confocal microscope (Carl Zeiss).
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4

Visualizing Acidic Autophagic Vesicles

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The acidic autophagic vesicles were visualized by acridine orange staining. Briefly, the MC3T3-E1 (5×104) cells were cultivated on glass chamber slides in α-MEM containing 10% FBS and SNP in the absence or presence of 3MA. At the end of the incubation period, cells were treated with 1 µg/ml acridine orange (Sigma) in serum-free medium for 15 min at 37℃. Subsequently, the cells were washed three times in PBS (acridine orange was removed) and examined under a Confocal Laser Scanning Microscope (Carl Zeiss, Germany). Depending on their acidity, autophagic lysosomes appeared as yellow-orange to bright-red fluorescent cytoplasmic vesicles, while nuclei were stained green.
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5

Visualization of Acidic Vesicles

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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).
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6

Evaluating Lysosomal V-ATPase Activity

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Lysosomal V-ATPase activity was assessed with acridine orange staining. acridine orange uptake represents lysosomal V-ATPase-driven pumping of hydrogen ions into the lysosomes36 (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 fluorescence10 (link),37 (link).
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7

Fluorescent Probes for Cellular Compartment Analysis

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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.
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8

Quantifying Apoptosis in Zebrafish Embryos

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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.
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9

Acridine Orange Assay for Osteoclast Acidification

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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.
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10

Cell Viability and Apoptosis Assay

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The cell viability was measured by WST-1 assay according to the manufacturer's protocol (Roche Applied Sciences). 5 × 103 cells were plated on 96-well plates and after overnight incubation treated with the indicated drugs for 72 hours. Cell viability defined as the absorbance in the treatment group compared to the control group was expressed as percent mean change ± SD (n = 4). Apoptosis was assessed using differential staining with acridine orange (green) (2 μg/ml; Sigma-Aldrich) incorporated by all cells and ethidium bromide (red) (2 μg/ml; Promega) incorporated only by apoptotic cells with compromised cell membrane. Quantification of apoptotic cells co-stained with acridine orange and ethidium bromide was performed on the images taken with confocal microscope Zeiss NLO710 as described [26 ]. Four replicate experiments were performed. Cells were counted in 5 independent random view fields.
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