Autophagy assay cell type - HT1080

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

LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Cells were collected and lysed in radioimmune precipitation assay buffer containing protease and phosphatase inhibitor mixtures at 4 °C for 30 min. Equal amounts of cellular proteins were analyzed by SDS-PAGE, followed by immunoblot. Anti-β-actin blot was used for the protein loading control. The heat shock procedure and GST pulldown were reported previously (29, 31). For starvation, NIH3T3 or U2OS cells were washed once with PBS and then incubated with EBSS for 30 min, 1 h, and 2 h. For rapamycin stimulation, cells were incubated with rapamycin or DMSO for 6 h or overnight.

Protocol tips
Cells were collected and lysed in radioimmune precipitation assay buffer containing protease and phosphatase inhibitor mixtures at 4 °C for 30 min. Equal amounts of cellular proteins were analyzed by SDS-PAGE, followed by immunoblot. Anti-β-actin blot was used for the protein loading control. The heat shock procedure and GST pulldown were reported previously (29, 31). For starvation, NIH3T3 or U2OS cells were washed once with PBS and then incubated with EBSS for 30 min, 1 h, and 2 h. For rapamycin stimulation, cells were incubated with rapamycin or DMSO for 6 h or overnight.

Protocol tips

Cells were collected and lysed in radioimmune precipitation assay buffer containing protease and phosphatase inhibitor mixtures at 4 °C for 30 min. Equal amounts of cellular proteins were analyzed by SDS-PAGE, followed by immunoblot. Anti-β-actin blot was used for the protein loading control. The heat shock procedure and GST pulldown were reported previously (29, 31). For starvation, NIH3T3 or U2OS cells were washed once with PBS and then incubated with EBSS for 30 min, 1 h, and 2 h. For rapamycin stimulation, cells were incubated with rapamycin or DMSO for 6 h or overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
	Incubate cells at 30 min at 37 °C after adding the dye	Incase of Low dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Protocol tips
Incubate cells at 30 min at 37 °C after adding the dye
Downstream tips
Incase of Low dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High 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

Dansylcadaverine

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
An autofluorescent dye that accumulates in the autophagic vacuoles.	50 mM MDC for 40 min at 37uC, washed twice with PBS, and then observed by a fluorescent microscope.

Upstream tips
An autofluorescent dye that accumulates in the autophagic vacuoles.
Protocol tips
50 mM MDC for 40 min at 37uC, washed twice with PBS, and then observed by a fluorescent microscope.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:200

Protocol tips
Dilute primary Ab at 1:200

Manufacturer protocol Publication protocol 1 Relevant paper

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