CYTO-ID® Autophagy detection kit

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Kit contains specific dye that selectively stains autophagic compartments.

Upstream tips
Kit contains specific dye that selectively stains autophagic compartments.

Publication protocol

Autophagic flux in human CC cell lines was analyzed by conversion of LC3-I to LC3-II and determined by western blotting using an anti-LC-3B antibody (Sigma-Aldrich) as described in [12]. In brief, after drug treatment, nuclear protein was isolated (Life Technologies, Carlsbad, CA, USA). Protein content was determined by applying the BCA Protein Assay Kit (Life Technologies). Equal amounts of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated overnight with primary antibodies against human LC3-II. After washing, membranes were incubated and developed with a horseradish peroxidase-conjugated secondary antibody (Life Technologies). β-actin served as an internal control.

Autophagic flux also was determined using the Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA), according to the manufacturer’s instructions described in [12]. This assay is based on a specific dye that selectively stains autophagic compartments. Autophagic flux is therefore quantified as accumulation of autophagic compartments in basic or activated conditions (starvation medium or PP242-incubation) after the blockage of autophagolysosomal degradation by CQ or ACH. Autophagic flux is calculated by subtracting the Cyto-ID mean fluorescence intensity (MFI) of the sample without CQ/ACH from the Cyto-ID MFI of the sample with CQ/ACH for each condition using the following 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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