Autophagy assay cell type - Gaucher macrophages

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

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	- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium	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
- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium
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

MAP LC3α/β Antibody (N-20): sc-16755

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	For Western Blot use starting dilution 1:100, (dilution range 1:100-1:1000)	- It is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies.

Protocol tips
For Western Blot use starting dilution 1:100, (dilution range 1:100-1:1000)
Downstream tips
- It is more specific, provides a stronger signal and eliminates lot to lot variation characteristic of most, if not all, polyclonal antibodies.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	1:1000 dilution for primary antibody

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