Cyto id autophagy detection kit

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen (Grand Island, NY, USA). Genistein (GEN), hydroxychloroquine, penicillin, streptomycin and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Immunoprecipitation (IP) lysis buffer, Dynabeads® Protein G, fluorescence isothiocyanate (FITC)-Annexin V and PI were purchased from Life Technologies Corporation (Carlsbad, CA, USA). Antibody against Bcl-xL was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against mTOR, p-mTOR, p62, Bax, β-tubulin, p-Akt, Akt, PARP, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were from Abcam (Cambridge, MA, USA). Beclin1, LC3, γ-H2AX, and caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Cyto-ID Autophagy Detection Kits were purchased from Enzo Life Sciences (Farmingdale, NY, USA), and NE-PER(R) Nuclear and Cytoplasmic Extraction Kits were from Thermo Scientific Pierce (Rockford, IL, USA).

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)].

For fluorescence microscopy, the Cyto-ID® Autophagy Detection Kit (Enzo Life Science) was used according to the manufacturer’s instructions. Stained cells from three biological replicates were visualized under an IX81 (MT10) fluorescent microscope. Green fluorescent autophagy-related organelles (pre-autophagosomes, autophagosomes, and autophagolysosomes) were quantified as green signal area per cell nucleus as advised by the manufacturer. Segmentation of these areas was based on 31 parameters assessing color, texture, and edge and was carried out in Ilastik, version 1.3.0 developed by the European Molecular Biology Laboratory, Heidelberg [58 (link)]. Classifiers trained for these parameters on a set of representative images were then applied to batch process multiple images as described in Ilastik’s user manual. Binary masks thus obtained were measured in FIJI [59 (link)] after applying a size filter to remove small size artifacts resulting from segmentation.
Transmission electron microscopy was performed as previously described [60 ].

To monitor the autophagy of live MKN-45 cells induced by BCA-M-PEG20, CYTO-ID® Autophagy detection kit was used (Enzo Life Sciences, Farmingdale, NY, USA). About 6000 MKN-45 cancer cells were seeded into confocal plates per well and incubated with or without BCA-M-PEG20 (0.58 µg/mL) for different durations. A positive control, Rapamycin (500 nM), was used in this assay. Then, cancer cells were stained with cyto-ID dye and Hoechst 33,342 following the instructions of the manufacturer and detected by a Leica TCS SPE confocal microscope.

The autophagy of cells was detected by Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, NY, USA). LC3II-positive punctate pattern was observed under confocal microscope (Carl Zeiss LSM 510 META Laser Confocal Microscope, Oberkochen, German). Number of autophagosomes was counted by using the ImageJ program (Version1.48u, Bethesda, USA).
Ultrathin sections (100 nm) were cut on an ultramicrotome, counterstained with 0.3% uranium acetate and lead nitrate, and examined by a transmission electron microscope (TEM) (H7700, Hitachi, Japan).

Both control and GMs macrophages were cultured in chamber slides. After the different treatments, cells were washed twice with 1X assay buffer and stained using a Cyto‐ID Autophagy Detection Kit (Enzo life science, Farmingdale, NY, USA), according to the manufacturer's instructions. Cells were imaged by confocal microscopy using a 40X oil DIC objective.