title: 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-Orange-stained 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.
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.
authors.forename: Marcos P
authors.initials: MP
authors.lastname: Thomé
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.|Faculdade de Medicina, UFRGS, 91501970, Porto Alegre, Brazil.
authors.forename: Eduardo C
authors.initials: EC
authors.lastname: Filippi-Chiela
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.
authors.forename: Emilly S
authors.initials: ES
authors.lastname: Villodre
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.
authors.forename: Celina B
authors.initials: CB
authors.lastname: Migliavaca
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.
authors.forename: Giovana R
authors.initials: GR
authors.lastname: Onzi
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil.|Departamento de Análises Clínicas, Universidade Federal do Paraná (UFPR), 80060000, Curitiba, Brazil.
authors.forename: Karina B
authors.initials: KB
authors.lastname: Felipe
authors.affiliation: Departamento de Biofísica, Universidade Federal do Rio Grande do Sul (UFRGS), 91501970, Porto Alegre, Brazil lenz@ufrgs.br.|Centro de Biotecnologia, UFRGS, 91501970, Porto Alegre, Brazil.
authors.forename: Guido
authors.identifier: 0000-0003-4077-6316
authors.initials: G
authors.lastname: Lenz
mesh_terms: D000143:Acids / Q000378:metabolism*; D000165:Acridine Orange / Q000378:metabolism*; D000818:Animals; D001343:Autophagy; D048429:Cell Size; D003584:Cytological Techniques / Q000379:methods*; D005434:Flow Cytometry; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D015388:Organelles / Q000378:metabolism*; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
chemical_list: D000143:Acids; D000165:Acridine Orange
keywords: Acidic organelle; Acridine Orange; Autolysosome; Autophagy; Flow cytometry; Single cell studies