Acridine orange (1 mg/mL) was added to untreated as well as treated cells for 15 min at 37 °C. Next, cells were washed twice with PBS. Images of cells were obtained using Olympus laser-scanning confocal microscope (FV-10 ASW 3.0 viewer image browser) at 450 nm and 593 nm for the detection of acidic vesicular organelles [3 (link)].
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2 protocols using fv 10 asw 3.0 viewer image browser
1
Detecting Acidic Vesicular Organelles
2
Quantification of Autophagy in Cells
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Acidic vesicular organelles (AVOs) were examined by microscopy and quantified using flow cytometry of acridine orange (AO) stained cells. Cells were isolated from tissues by collagenase-hyaluronidase digestion and stained with AO (1µg/ml) by incubation at 37°C for 15 minutes in dark. Following subsequent PBS washes, the cells were observed under Olympus laser-scanning confocal microscope (FV-10 ASW 3.0 viewer image browser) at 450 and 593 nm to determine AVOs. AO stained cells were also analyzed in a flow cytometer (red channel, 630 nm) using the BD Accuri (BD Biosciences; USA) to estimate the percentage of cells undergoing autophagy following ART treatment (36 (link)).
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