Measurement of Autophagic Activity

Cells were incubated in HBSS containing CYTO-ID (Enzo Life Sciences, Farmingdale, NY) and Hoechst (0.1μg/mL) (Immunochemistry Technologies) for 40 min. Thereafter, autophagosomes and autolysosomes (AV/AL) fluorescence was measured using a Biotek FLx800 plate reader (excitation 485 nm/emission 528 nm). Values were normalized to Hoechst fluorescence. CYTO-ID staining in cells was observed with a deconvolution microscopy system (Delta Vision imaging system, GE Healthcare Life-Sciences). The use of CYTO-ID to measure autophagy has been validated in previous reports (Guo et al 2015 (link); Chan et al. 2012 (link)).

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Liuzzi J.P., Narayanan V., Doan H, & Yoo C. (2018). Effect of zinc intake on hepatic autophagy during acute alcohol intoxication. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 31(2), 217-232.

Publication 2018

CYTO-ID Hoechst FLx800 plate reader Delta Vision imaging system

Autolysosomes Autophagosomes Autophagy Cells Fluorescence Hbss Microscopy

Corresponding Organization : Florida International University

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Variable analysis

independent variables

Incubation of cells in HBSS containing CYTO-ID and Hoechst (0.1μg/mL)

dependent variables

Autophagosomes and autolysosomes (AV/AL) fluorescence measured using a Biotek FLx800 plate reader (excitation 485 nm/emission 528 nm)
Cyto-ID staining in cells observed with a deconvolution microscopy system (Delta Vision imaging system, GE Healthcare Life-Sciences)

control variables

Incubation time (40 min)
Concentrations of CYTO-ID and Hoechst (0.1μg/mL)

controls

The use of Cyto-ID to measure autophagy has been validated in previous reports (Guo et al 2015; Chan et al. 2012)

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