CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	Incubate hepatocytes in Cyto-ID green solution dye in dark for 30 min at 37 degreeC	Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experimen

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Incubate hepatocytes in Cyto-ID green solution dye in dark for 30 min at 37 degreeC
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experimen

Publication protocol

Primary hepatocyte isolation was performed as we previously described.38 The isolated cells were plated on dishes (3 × 106 cells/6-cm dish), six-well plates (2 × 105 cells/well) or 24-well plates (8 × 104 cells/well). To simulate H/R in vitro, hepatocytes were cultured for 4 h at 37 °C in a modular incubator chamber (Biospherix, Lacona, NY, USA) gassed with 5% CO2, 90% N2 and 5% O2 for 4 h. For reoxygenation, hepatocytes were returned to the normoxic incubator for 2 h. To assess the cell injury, LDH was measured by LDH Release Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. Mitochondrial transmembrane potential in hepatocytes was assessed by ΔΨm assay kit with JC-1 (Beyotime) according to the manufacturer’s protocols. The decrease of ΔΨm was assessed by transition from JC-1 aggregates (Red) to JC-1 monomer (Green) in cells which indicating mitochondrial dysfunction and early cell death. Mitochondrial generation of superoxide was stained with MitoSOX Red (Invitrogen, Waltham, MA, USA). The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmington, NY, USA) was used to identify the autophagosomes of the hepatocytes. In brief, after H/R injury, the hepatocytes were incubated in Cyto-ID Green stain solution in the dark for 30 min at 37 °C. Then analyze the stained cells by fluorescence microscope.

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Manufacturer protocol

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