Autophagy assay cell type - Mouse embryonic fibroblasts

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

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 3 matching solutions for this experiment

LC3B Antibody Kit for Autophagy

Thermo Fisher Scientific

Upstream tips
Cells were lysed at 4°C in buffer containing 1% SDS, 10 mM Tris-HCl pH 7.4, supplemented with protease inhibitors (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Sigma Chemical Co., St. Louis, MO). Viscosity of the samples was reduced by brief sonication. 40 µg of protein (Bio-Rad Protein Assay) were boiled for 5 min in Tris-glycine-SDS sample buffer (Invitrogen) and heated at 70°C for 10 min, then separated by SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA).
Protocol tips
After blocking nonspecific binding sites for 1 h with 5% milk in TPBS (phosphate-buffered saline, Tween20 0.1%), membranes were incubated overnight with primary antibody. After three washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit (1∶10,000 dilution), goat anti-rabbit (10,000 dilution), or anti-mouse (1∶5,000 dilution) antibody (Amersham Biosciences, Piscataway, NJ) for 1 h and then washed three times in TPBS.
Downstream tips
Immunoblots were revealed using enhanced chemiluminescence detection kit (Pierce) by autoradiography.
Protocol tips
Grow the cells in EBSS medium (amino acid depleted) medium to induce autophagy. Immunofluorescence staining to visualize the autophagosome.
Downstream tips
Incase of Low CYTO-ID 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 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
Antibody is recommended for western blotting and immunofluorescence staining
Protocol tips
Methyl-beta cyclodextrin treatment in breast cancer cell lines induces autophagic flux that can be detected with Anti -LC3B antibody by western blot.
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!

Outsource your experiment

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

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms