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	Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min	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
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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

Flow cytometry analysis
Experiments were performed in BD FACS CaliburTM. The HCE cells were trypsinized and 10,000 cells were recorded for analysis under both normoxic and oxidatively stressed conditions on Day 3 and 7. Quantification of ROS (2’,7’–dichlorofluorescin diacetate [DCFDA], Life Technologies, FL-1), autophagosomes (CYTO-ID Autophagy Detection Kit, Enzo Lifesciences, FL-1) and lysosomal content (LysoTracker® Red DND-99, Life Technologies, FL-3) were assessed as per the manufacture’s protocol. Data was analyzed with the Cell Quest Pro Software (Version 6.0).

Fluorescence imaging
For imaging, the HCE cells were plated on 8-chamber slides (SPL Life Sciences) at a density of 100 cells/chamber and subjected to normoxic and oxidative stress conditions until Day 7. For monitoring autophagosomes, HCE cells were incubated with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min. Lysosomal content was monitored using LysoTracker® Red DND-99 (500nM) for 15 mins at 37°C (Life Technologies, MA, USA). Cells were fixed with 4% paraformaldehyde (pH7.2), washed and examined under fluorescent microscope (Zeiss, Oberkochen, Germany) and the images were captured using Axiocam, a ZEN software. Numbers of cells counted for red and yellow fluorescence were more than 25 for each condition.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - HCE using CYTO-ID® Autophagy detection kit from Enzo Life Sciences.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - HCE using CYTO-ID® Autophagy detection kit from Enzo Life Sciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.