CYTO-ID® Autophagy detection kit

Autophagy assay cell type - PANC-1

Experiment
Autophagy assay cell type - PANC-1
Product
CYTO-ID® Autophagy detection kit from Enzo Life Sciences
Manufacturer
Enzo Life Sciences

Protocol tips

Protocol tips
Stain the cells with CYTO-ID for 30 min at 37°C in the dark

Publication protocol

Quantification of cellular autophagy
To examine the effect of PLB on autophagy in PANC-1 and BxPC-3 cells, cellular autophagy was detected using flow cytometry as described previously.21 Briefly, PANC-1 and BxPC-3 cells were seeded in 60 mm Petri dishes. After cells were seeded for 24 hours, the cells reached ~75% confluence and then treated with fresh medium alone, control vehicle alone (0.05% DMSO, v/v), or PLB (0.1, 1, and 5 μM) for 24 hours. In separate experiments, PANC-1 and BxPC-3 cells were treated with 5 μM PLB for 4, 8, 12, 24, 48, and 72 hours. Following the PLB treatment, cells were resuspended in 250 μL of phenol red-free culture medium containing 5% FBS, and 250 μL of the diluted Cyto-ID®Green stain solution was added to each sample and mixed well. Cells were incubated for 30 minutes at 37°C in the dark and then collected by centrifugation at 250× g. The cell pellet was washed with 1× assay buffer in the Cyto-ID® autophagy detection kit and resuspended in 500 μL fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer (Becton Dickinson Immunocytometry Systems).

Confocal fluorescence microscopy
In order to further detect the cellular autophagy level, the cellular autophagy level was examined using confocal fluorescence microscopy. Briefly, PANC-1 and BxPC-3 cells were seeded into an 8-well chamber slide. The cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours. In separate experiments, cells were treated with 5 μM of PLB for 4, 8, 12, 24, 48, and 72 hours. After the PLB treatment, the cells were washed with 1× assay buffer in the Cyto-ID® autophagy detection kit, following by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After the incubation, the cells were washed with 1× assay buffer to remove detection reagent, and the cells were then examined using a Leica TCS SP2 laser scanning confocal microscopy (Wetzlar, Germany) using a standard fluorescein isothiocyanate filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

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

Reviews

CYTO-ID® Autophagy detection kit from Enzo Life Sciences has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - PANC-1 using CYTO-ID® Autophagy detection kit from Enzo Life Sciences.

Paper title
Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells
Full paper
Login or join for free to view the full paper.

Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - PANC-1 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.

We haven't found any additional videos for this experiment / product combination yet.

Become shareholder Discussions About us Contact Privacy Terms