Autophagy assay cell type - MCF7

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 uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	Incubate cells for 30 minutes at 37°C in the dark, wash with 1× assay buffer, and resuspend in 500 μL fresh 1× assay buffer. Subject the cells to flow cytometric analysis using the green (FL1) channel.	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 uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Incubate cells for 30 minutes at 37°C in the dark, wash with 1× assay buffer, and resuspend in 500 μL fresh 1× assay buffer. Subject the cells to flow cytometric analysis using the green (FL1) channel.
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

LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
LC3A/B (D3U4C) XP Rabbit mAb recognizes endogenous levels of total LC3A and LC3B proteins.	1:1000 dilution of primary antibody

Upstream tips
LC3A/B (D3U4C) XP Rabbit mAb recognizes endogenous levels of total LC3A and LC3B proteins.
Protocol tips
1:1000 dilution of primary antibody

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A/B Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:300

Protocol tips
Dilute primary Ab at 1:300

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 Antibody

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:500

Protocol tips
Dilute primary Ab at 1:500

Manufacturer protocol Publication protocol 1 Relevant paper

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