Autophagy assay cell type - MLO-Y4

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

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
This kit helps to monitor Autophagy in live cells by Flow Cytometry, fluorescence microscopy and microplate reader.	MLO-Y4 cells to stain with Cyto-ID to visualize autophagosome following Dexamethasone treatment	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 helps to monitor Autophagy in live cells by Flow Cytometry, fluorescence microscopy and microplate reader.
Protocol tips
MLO-Y4 cells to stain with Cyto-ID to visualize autophagosome following Dexamethasone treatment
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

The Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Membranes were blocked with 5% skim milk in TBS-Tween (TBST) for 1hr and then incubate with primary antibodies diluted in 1% (w/v) skim milk in TBST for 2hrs shaking at RT or overnight shaking at 4°C

Protocol tips
Membranes were blocked with 5% skim milk in TBS-Tween (TBST) for 1hr and then incubate with primary antibodies diluted in 1% (w/v) skim milk in TBST for 2hrs shaking at RT or overnight shaking at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 (Rat) pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
	Primary antibody dilution 1:1000

Protocol tips
Primary antibody dilution 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 (Rat) pAb

MBL international corporation

Upstream tips	Protocol tips	Downstream tips
	Primary antibody dilution 1:1000

Protocol tips
Primary antibody dilution 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

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