Ratiometric analysis of acridine orange staining in the study of acidic organelles and autophagy

Abstract

Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orangestained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.

Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

“…Meanwhile, AO could be protonated and trapped in acidic vesicular organelles such as autolysosomes and fluorescence red in a concentration-dependent manner. Taking the advantages of quickness and high reliability, AO staining was usually used to assess the volume of autolysosomes [41]. Cells were cultured at 37 °C in a 6-well plate with respective stimulation in different groups for 24 h. After rinsing with phosphate buffered saline (PBS), 1 mL of AO dyeing working solution (0.1 mg/mL, final concentration, in the dark) was added to the plate for 3 min.…”

High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

“…At high concentrations, AO dimerizes, causing a metachromatic shift from green to red (Figure 1), which can be measured for studying late-stage autophagy. Thomé et al can be referenced for detailed chemical and concentration changes [10]. AO can be assessed using fluorescence microscopy or flow cytometry, but these approaches suffer from the limitations described above (Table 1).…”

“…The monochromator setting was used to measure excitation/emission wavelengths of 500/526 (green) to assess intensity of unprotonated, diffuse AO staining DNA (non-autophagic staining), and 460/650 (red) to assess intensity of dimerized, protonated AO concentrated in AVOs (autophagic staining) [10]. The MiniMax setting was used to count the number of cells per well as we have previously described [12].…”

“…The MiniMax setting was used to count the number of cells per well as we have previously described [12]. The red intensity signal per well was divided by the green intensity or the cell count to assess the level of autophagy and to assess the efficacy of the two normalization methods: controlling to green intensity as recommended by Thome et al versus controlling to cell count as previously done by Fowler et al [10,12]. The statistical analysis performed on the IF data was performed on the AO assay recorded values.…”

“…In the Baf- group, the increase in red cytoplasmic intensity in conjunction with a decrease in green nuclear intensity with increasing doses of PP242 reflects a PP242 dose-dependent increase in basal autophagy [10]. In the Baf+ group, no significant differences are observed between groups, since Baf inhibits late-stage autophagy (i.e., accumulation of AVOs).…”