CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Mouse Aortic Endothelial cells (MAECs) can be induced to stain with Cyto-ID dye by treating them with PM2.5 . Stained cells can be used to visualize either by Flow Cytometry or Confocal microscopy.	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.

Protocol tips
Mouse Aortic Endothelial cells (MAECs) can be induced to stain with Cyto-ID dye by treating them with PM2.5 . Stained cells can be used to visualize either by Flow Cytometry or Confocal microscopy.
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.

Publication protocol

Cellular autophagy was monitored by Western blot analysis of specific key proteins (microtubule-associated protein 1A/1B light-chain 3B (LC3B) and Beclin-1), confocal microscopy (LSM510 META; Zeiss, Oberkochen, Germany), and flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). The presence of autophagosomes in PM2.5-induced MAECs was assessed using the Cyto-ID autophagy detection kit (175-0050; Enzo Life Sciences, Farmingdale, NY, USA), which was used to monitor specific autophagic fluorescence signals by confocal microscopy, or to quantitatively measure autophagic fluorescence intensity by flow cytometric analysis. For the immunoblot assay, induction of cellular autophagy in response to PM2.5 exposure was determined according to an increase in the endogenous LC3B-II:LC3B-I ratio and upregulation of Beclin-1 expression. Autophagic flux was assessed by comparing the extent of LC3B-II accumulation following PM2.5 treatment in the presence or absence of concomitant use of CQ, an inhibitor that disrupts lysosomal function.

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

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