Autophagy assay cell type - OE21

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 3 matching solutions for this experiment

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.	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 experiment.

Upstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.
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 experiment.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3 Antibody (APG8B) (N-term)

Abgent

Upstream tips	Protocol tips	Downstream tips
This LC3 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 1-30 amino acids from the N-terminal region of human LC3.	Dilute primary Ab at 1:100

Upstream tips
This LC3 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 1-30 amino acids from the N-terminal region of human LC3.
Protocol tips
Dilute primary Ab at 1:100

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 (Rat) pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
This antibody reacts with LC3-I (18kDa) and LC3-II (16 kDa) on Western blotting	1:500 dilution of primary antibody

Upstream tips
This antibody reacts with LC3-I (18kDa) and LC3-II (16 kDa) on Western blotting
Protocol tips
1:500 dilution of primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

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