Autophagy assay cell type - MG-63

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

Upstream tips
250 µL of the diluted Cyto-ID® green stain solution was added to each sample and mixed well instead of 100ul
Protocol tips
Incubate for 30 minutes at 37°C in the dark
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
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in pre-cooled RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.25% sodium deoxycholate, 0.1% NP-40, 1 mM phenylmethysulfonyl fluoride (PMSF), 50 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM NaF, 5 mM EDTA, 5 mM EGTA and a protease inhibitor cocktail.
Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4ºC
Dansylcadaverine

Sigma-Aldrich

Protocol tips
If no detectable levels of autophagy in testing samples make sure that Autophagy Reagent A is added 2 hours prior to cell acquisition

Label cells with 0.05 mM MDC in PBS at 37°C for 10 min
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