Autophagy assay cell type - Mesenchymal stromal cells

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 2 matching solutions for this experiment

Protocol tips
Autophagosomes were detected with an autofluorescent compound monodansylcadaverine (MDC), using a Cell Meter Autophagy kit (AAT Bioquest, Inc.). Briefly, BMSCs were seeded on glass coverslips in a 24-wells plate at a density of 3 x 104/well. After incubation, BMSCs were stained with 1 x MDC solution and incubated at 37°C for 1 hour. Cells were washed three times and examined under a confocal microscope (Ex = 330 nm).
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 experimen
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