Autophagy assay cell type - GL15

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.

Start discussion

No discussions found

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

Acridine Orange hemi(zinc chloride) salt

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Intercalates between DNA/RNA and used to analyze mitochondria and Lysosomal content by flow cytometry.	Stain the cells with 2 μg/ml acridine orange (AO) in phosphate buffered saline for 15 min at 37 °C and immediately analyze with a fluorescence microscope

Upstream tips
Intercalates between DNA/RNA and used to analyze mitochondria and Lysosomal content by flow cytometry.
Protocol tips
Stain the cells with 2 μg/ml acridine orange (AO) in phosphate buffered saline for 15 min at 37 °C and immediately analyze with a fluorescence microscope

Manufacturer protocol Publication protocol 1 Relevant paper

mTOR Inhibitor XI, Torin1 - Calbiochem

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Highly potent inhibitor for mTOR and DNA-PK while inhibits PI3-K only at much higher concentration	Culture the cells in DMEM medium containing 250nM Torin1

Upstream tips
Highly potent inhibitor for mTOR and DNA-PK while inhibits PI3-K only at much higher concentration
Protocol tips
Culture the cells in DMEM medium containing 250nM Torin1

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A/B (D3U4C) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Gives distinct bands corresponding to LC3I and LC3II	1:1000 dilution for primary antibody

Upstream tips
Gives distinct bands corresponding to LC3I and LC3II
Protocol tips
1:1000 dilution for primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Fill out your contact details and receive price quotes in your Inbox