Autophagy assay cell type - Human fetal osteoblastic (hFOB) 1.19

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 4 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	Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min	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
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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

Autophagy Inhibitor, 3-MA

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Use 1mM concentration of 3-MA for in vitro treatment of cells

Protocol tips
Use 1mM concentration of 3-MA for in vitro treatment of cells

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B (D11) XP® Rabbit mAb

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

Beclin-1 (D40C5) Rabbit mAb

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