CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For 100 μL of whole blood use 0.25 μL/mL of Cyto-ID Green Autophagy Detection Reagent for staining.	Stain for 30 min at RT in dark.	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
For 100 μL of whole blood use 0.25 μL/mL of Cyto-ID Green Autophagy Detection Reagent for staining.
Protocol tips
Stain for 30 min at RT in dark.
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

Fluorescence of autophagosomotropic dye, Cyto-ID, in circulating immune cells was measured using Cyto-ID™ Autophagy Detection Kit (Enzo Life Sciences, PA, USA) according to the manufacturer’s protocol and the described technique [26, 43-44]. Briefly, 100 μL of whole blood was stained with 0.25 μL/mL of Cyto-ID Green Autophagy Detection Reagent (Enzo Life Sciences, PA, USA) and 20 μL of Phycoerythrin-Cyanin 5 (PC5)-conjugated CD45-specific monoclonal antibody (mAb) (Beckman Coulter, Indianapolis IN, USA). After incubation for 30 min in the dark at room temperature, cells were reacted with OptiLyse Solution (Beckman Coulter, Indianapolis IN, USA) for 10 min to lyse red blood cells. After PBS washing, cells were analyzed by flow cytometry (Beckman Coulter, Brea, CA, USA). Monocytes, lymphocytes, and granulocytes were gated by CD45+/side scatters. At least 5 x 104 total cells from each sample were analyzed. The gated lymphocytes, monocytes, and granulocytes were verified as follows: 100 μL samples of whole blood stained with 20 μL of fluorescein isothiocyanate (FITC)-conjugated CD3-specific mAb (Beckman Coulter, USA), 20 μL of PC5-conjugated CD14-specific mAb, and 20 μL of FITC-conjugated CD66b-specific mAb, respectively, with 20 μL of PC5-conjugated CD45-specific mAb separately for 15 min at room temperature. Data were expressed as the MFI of Cyto-ID.

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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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