Flowcellect autophagy lc3 antibody based assay kit

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 (link), 35 (link)]. 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 (link)].

The leukemic cells were treated with IM-7 at the IC₅₀ concentration for 24 and 48 h. The LC3-II level was measured using FlowCellect™ Autophagy LC3 Antibody-based Assay kit (Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, at 30 min before the end of incubation time, 10 μl of diluted reagent A was added followed by incubation for 30 min at 37 °C. Then, the cell pellet was resuspended in 100 μl of reagent B followed by immediate spinning. The cells were stained with anti-LC3/FITC antibody for 30 min in the dark and analyzed by flow cytometer. The LC3-II level was calculated from the mean fluorescence intensity (MFI) by the following equation: relative LC3-II level = MFI (Treatment)/MFI (Control).