Autophagy assay cell type - MT-2 (human T cell leukaemia)

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 5 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	Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.	Incase of Low CYTO-ID 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 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
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.
Downstream tips
Incase of Low CYTO-ID 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 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

FlowCellect Autophagy LC3 antibody based kit

Millipore

Upstream tips	Protocol tips	Downstream tips
	To facilitate monitoring of lipidated LC3-II, pre-incubate the cells for 30 min with the autophagy flux inhibitor provided in the kit

Protocol tips
To facilitate monitoring of lipidated LC3-II, pre-incubate the cells for 30 min with the autophagy flux inhibitor provided in the kit

Manufacturer protocol Publication protocol 1 Relevant paper

Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Protocol tips
For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Manufacturer protocol Publication protocol 1 Relevant paper

Atg5 (D5F5U) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Protocol tips
For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Manufacturer protocol Publication protocol 1 Relevant paper

Atg12 (D88H11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Protocol tips
For WB dilute primary Ab at 1:1000 and for Immunoprecipitation at 1:100

Manufacturer protocol Publication protocol 1 Relevant paper

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