FlowCellect Autophagy LC3 antibody based kit

Protocol tips

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	To facilitate monitoring of lipidated LC3-II, pre-incubate the cells for 30 min with the autophagy flux inhibitor provided in the kit

Protocol tips
To facilitate monitoring of lipidated LC3-II, pre-incubate the cells for 30 min with the autophagy flux inhibitor provided in the kit

Publication protocol

Autophagy was evaluated using the FlowCellect™ Autophagy LC3 Antibody-Based Assay Kit (Merck Millipore) according to the manufacturer’s instructions [34]. Briefly, discrimination between cytosolic and autophagosome-associated LC3 was achieved by monitoring translocation of LC3 using flow cytometry. As autophagy is a constitutive cellular degradation process, pretreatment of 72-h culture samples with a lysosomal inhibitor was required for 30 min prior to treatment with anti-LC3 conjugated to fluorescein isothiocyanate isomer-I (FITC) to prevent lysosomal degradation of LC3. After treatment, quantification of FITC fluorescence can then be performed. Cytosolic and autophagosomic populations are differentiated by washing cells to remove cytosolic LC3-I and retaining only membrane-bound LC3-II prior to staining.

The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions [17, 35]. Autophagy analysis was performed by incubating cells with bafilomycin A1 for 30 min at 37°C, washing cells, and analyzing fluorescence by flow cytometry using a Cell Analyzer EC800 (Sony, Tokyo, Japan) and fluorescence microscopy using a BZ-X710 fluorescence microscope (KEYENCE, Itasca, IL, USA), as previously described [14].

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