CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry		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
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

Publication protocol

Autophagic compartments (pre-autophagosomes, autophagosomes, and autophago-lysosomes) represent intermediate components of a dynamic degradation process and their total amount at a particular time point is determined by the dynamics of their creation and degradation [23]. We conducted studies that measure the autophagic flux according to recent guidelines [23]. Autophagic flux refers to the entire process of autophagy including cargo delivery and subsequent autolysosomal degradation. Measurement of autophagic flux allows the discrimination between induction and late inhibition of autophagosome maturation as both of them are characterized with an increased presence of autophagosomes [23], [24]. Conversion of GFP-LC3-I to GFP-LC3-II was determined by western blotting using anti-LC-3B antibody (Sigma-Aldrich) [23]. Autophagic flux was assessed as accumulation of undegraded GFP-LC3-II after blocking autophagosomal degradation using ACH. Densitometry LC3-II bands was performed using LabImage1D Software (Kapelan Bio-Imaging Solutions) and normalized to the optical density (OD) of actin [24]. Additionally, autophagic flux in HCC cell lines was analyzed by detection of changes in total cellular GFP-LC3 or mCherry-GFP-LC3 signal using flow-cytometry as previously described [20]. Briefly, increased autophagic flux corresponds to a progressive delivery of GFP-LC3 to autolysosomes for degradation and signal disappearance. Furthermore, inhibited autophagic flux translates into reduced GFP-LC3 disappearance and due to the constitutive cellular production is detected as an increased total cellular signal [20]. Analysis was made on LSR-II (BD Biosciences). Autophagic flux also was determined using Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) according to manufacturer's instructions as previously described [25]. This assay is based on a specific dye that selectively stains autophagic compartments. Autophagic flux thus is quantified as accumulation of autophagic compartments in basic or activated conditions (HBSS- or PP242-incubation) after the blockage of autophagolysosomal degradation by CQ or ACH. It is calculated by subtracting the Cyto-ID MFI value of the sample without CQ/ACH from the Cyto-ID MFI value of the sample with CQ/ACH for each condition using the formula: ΔMFI Cyto-ID = MFI Cyto-ID (+CQ/ACH) - MFI Cyto-ID (-CQ/ACH)

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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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