Autophagy assay cell type - Hepatocytes

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 4 matching solutions for this experiment

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
Protocol tips

Autophagic flux was assessed by increase in GFP-LC3 puncta or Western blot analysis of LC3 in hepatocytes after treatment with bafilomycin (50 nm, Sigma) for 1 h or transfecting hepatocytes with the GFP-RFP-LC3 plasmid. 24 h after transfection, cells were imaged with a Zeiss LSM 510 laser-scanning confocal microscope using a ×63 oil lens. The numbers of GFP and RFP double-positive (early autophagic vacuoles) and RFP-only (late autophagic vacuoles) puncta were counted for each cell.
Protocol tips
Dilute primary Abbetween 1:500- 1:2000
LC3B Antibody #2775

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1000
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