Autophagy assay cell type - Keratinocytes

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 3 matching solutions for this experiment

LC3B Antibody

Novus Biologicals

Upstream tips	Protocol tips	Downstream tips
	Western analysis and cytokine ELISAs (for TNF-α and IL-6) were performed, as described previously (17). NF-κB luciferase assays were also performed, as described previously (17). Briefly, cells were cotransfected with a NF-κB–luc construct and siRNA (for HaCaT cells) or transduced with Ad5HSV-NF-κB-luc. Following stimulation with MALP-2 (100 ng/ml) for 24 h, cells were lysed and their luciferase activity was analyzed using the Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.

Protocol tips
Western analysis and cytokine ELISAs (for TNF-α and IL-6) were performed, as described previously (17). NF-κB luciferase assays were also performed, as described previously (17). Briefly, cells were cotransfected with a NF-κB–luc construct and siRNA (for HaCaT cells) or transduced with Ad5HSV-NF-κB-luc. Following stimulation with MALP-2 (100 ng/ml) for 24 h, cells were lysed and their luciferase activity was analyzed using the Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.

Protocol tips

Western analysis and cytokine ELISAs (for TNF-α and IL-6) were performed, as described previously (17). NF-κB luciferase assays were also performed, as described previously (17). Briefly, cells were cotransfected with a NF-κB–luc construct and siRNA (for HaCaT cells) or transduced with Ad5HSV-NF-κB-luc. Following stimulation with MALP-2 (100 ng/ml) for 24 h, cells were lysed and their luciferase activity was analyzed using the Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.

Manufacturer protocol Publication protocol 1 Relevant paper

SQSTM1 Antibody (H-290)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Protocol tips
Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000)

Manufacturer protocol Publication protocol 1 Relevant paper

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
	Staining for 20 min at 37degreec C.

Protocol tips
Staining for 20 min at 37degreec C.

Manufacturer protocol Publication protocol 1 Relevant paper

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