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	- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium	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
Protocol tips
- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium
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

Quantification of cellular autophagy:
Hep3B cells were treated with ALS at 0.1, 1, and 5 µM for 24 h, or treated with 1 µM ALS for 4, 8, 12, 24, 36 and 60 h, then subject to flow cytometry for analyzing the intracellular autophagy level. The cells were dyed with green detection and Hoechst 33342 nuclear stain reagent contained in the Cyto-ID® autophagy detection kit (No. ENZ-51031-K200) according to the manufacturer’s instructions. The cells were analyzed using the green (FL1) channel of a flow cytometer (Becton Dickinson Immunocytometry Systems).

Confocal fluorescence microscopy examination:
Hep3B cells were treated with ALS at 0.1, 1, and 5 µM for 24 h, then subjected to confocal fluorescence microscopy for detecting the intracellular autophagy level. The cells were processed with the Cyto-ID® autophagy detection kit (No. ENZ-51031-K200) according to the manufacturer’s instructions. The cells were examined using a Leica TCS SP2 laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a standard FITC filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

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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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