title: Rapid screening of potential autophagic inductor agents using mammalian cell lines.
abstract: Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.
authors: Department of Biochemistry, University of São Paulo, São Paulo, Brazil. wkerllenmartins@gmail.com
authors: Waleska K
authors: WK
authors: Martins
authors: Divinomar
authors: D
authors: Severino
authors: Cleidiane
authors: C
authors: Souza
authors: Beatriz S
authors: BS
authors: Stolf
authors: Maurício S
authors: MS
authors: Baptista
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D055808:Drug Discovery / Q000379:methods*; D005840:Gentian Violet / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D009499:Neutral Red / Q000494:pharmacology; D015203:Reproducibility of Results; D018709:Statistics, Nonparametric; D019311:Staurosporine / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D007202:Indicators and Reagents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009499:Neutral Red; D006861:Hydrogen Peroxide; C022616:thiazolyl blue; D019311:Staurosporine; D005840:Gentian Violet