Autophagy assay cell type - MDA-MB-231

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 4 matching solutions for this experiment

Protocol tips
Cells were resuspended in 250 μL of phenol red-free culture medium (Thermo Fisher Scientific Inc.; No: 1294895) containing 5% FBS, and 250 μL of the diluted Cyto-ID® Green stain solution was added to each sample and mixed well. Cyto-ID® Green stain is a cationic amphiphilic tracer that selectively labels autophagic vacuoles in cells. Cells were incubated for 30 minutes at 37°C in the dark, collected by centrifugation, washed with 1× assay buffer, and resuspended in 500 μL fresh 1× assay buffer. Cells were subjected to flow cytometric analysis using the green (FL1) channel. The flow cytometer collected 10,000 events.
Upstream tips
Lyse cells in in RIPA lysis buffer containing protease inhibitors
Protocol tips
Dilute primary Ab at 1:1,000 for immunoblotting
SQSTM1/p62 (D5L7G) Mouse mAb #88588

Cell Signaling Technology

Protocol tips
For WB , dilute primary Ab at 1:1000 and for immunoflourence at 1:800.

Incubate at 4°C overnight for WB
Protocol tips
Incubate antibody at 4°C for
30 minutes in the dark
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