Cyto-ID^® Autophagy detection kit

A No-transfection, Quantitative Assay for Monitoring Autophagy in Live Cells



ENZ-51031-K200	1 Kit	365.00 USD

No transfection required
Proprietary dye includes titratable moieties specific for selectively staining autophagic vesicles
Protocol validated with known inhibitors and activators of autophagic activity
Rapidly quantifies autophagy in native heterogeneous cell populations
Eliminates need for time and effort-consuming transfection efficiency validation required with LC3-GFP transfection
Selective and comprehensive staining, allows measurement and differentiation between autophagic flux and autophagolysosome accumulation
Negligible staining of lysosomes reduces background seen with other dyes
Facilitates high-throughput screening of activators and inhibitors of autophagy

Cyto-ID^® Autophagy Detection Kit measures autophagic vacuoles and monitors autophagic flux in live cells using a novel dye that selectively labels autophagic vacuoles. The dye has been optimized through the identification of titratable functional moieties that allow for minimal staining of lysosomes while exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). The assay offers a rapid and quantitative approach to monitoring autophagy in live cells without the need for cell transfection.

Mechanism of Action
The probe is a cationic amphiphilic tracer (CAT) dye that rapidly partitions into cells in a similar manner as drugs that induce phospholipidosis.

Profile autophagy without transfection. Figure 1A: CHO cells stably expressing GFP-LC3 transfected cell lines results in relatively poor baseline separation of control-vs-starved cell populations, making quantification of autophagy difficult. Figure adapted from Shvets E, Fass E, Elazar Z. Figure 1B: The Cyto-ID® Autophagy Detection Kit specifically labels autophagic vacuoles independent of LC3 protein and eliminates the need for transfection. HeLa cells were subjected to starvation and recovery and then labeled with Cyto-ID® Green detection reagent. The dye enables clear detection and quantification of autophagic and pre-autophagic vacuoles that directly correlates to induction of autophagy.

Figure 2: Visualization of autophagic accumulation and autophagic flux. Autophagic vacuole accumulation and flux are both detected by Cyto-ID® Autophagy Green dye as observed by fluorescence microscopy. HeLa cells were mock-induced with 0.2% DMSO (A ) or induced with 100 uM Clonidine hydrochloride (B), 5 uM Loperamide hydrochloride (C ) or 1 uM PP242 hydrate (D) for 12 hours at 37°C. After treatment, cells were incubated with Cyto-ID® Green Detection reagent for 10 min at 37°C and then washed with assay buffer. Nuclei were counter-stained in blue with Hoechst 33342 dye.

Figure 3. Time-saving, rapid and comprehensive labeling of autophagic vacuoles without transfection. For the purpose of demonstrating advantages of Cyto-ID® Green detection reagent, HeLa cells were first transfected with RFP-LC3 expression vector, treated with 10 µM Tamoxifen overnight, then stained with Cyto-ID® Green detection reagent. Unlike overnight transfection-based assays, the Cyto-ID® Green detection reagent approach labels 100% of cells in 15-30 minutes. Panel A: Green signal indicating Cyto-ID® Green staining of autophagic vesicles; Panel B: RFP-LC3 expression (red) in a subset of successfully transfected cells; Panel C: Composite image, showing Cyto-ID® Green dye-labeled vesicles co-localize with LC3, a specific marker of autophagosomes.

Figure 4: Eliminate background resulting from non-specific lysosomal staining. Cyto-ID® Green dye eliminates background staining of lysosomes seen with other lysosomotrophic dye-based assays that utilize monodansylcadaverine (MDC) (bottom panel). The Cyto-ID® Autophagy kit eliminates the need for a 350 nm UV laser for live cell analysis, and is compatible for use with Hoechst dyes for co-labeling in microscopy applications.

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Product Specification

Quantity:	200 flow cytometry assays or 125 microscopy assays.

Quality Control:	A sample from each lot of Cyto-ID^® Autophagy detection kit is used to stain Jurkat cells and analyzed by flow cytometry, using the procedures described in the user manual. The MAF values for the samples were greater than 25. The percentage of viable cells in the control samples is > 90% and > 80% in the treated samples.

Kit/Set Contains:	Cyto-ID^® Green Detection Reagent, 25 µl Hoechst 33342 Nuclear Stain, 50 µl Autophagy Inducer (Rapamycin, 12.5 nmol) 10X Assay Buffer, 30 ml

Application:	The Cyto-ID^® Autophagy detection kit provides a rapid, specific and quantitative approach for monitoring autophagy in live cells by fluorescence microscopy and flow cytometry.

Short Term Storage:	-20°C

Long Term Storage:	-80°C

Use/Stability:	With proper storage, the kit components are stable for one year from date of receipt.

Handling:	Avoid freeze/thaw cycles. Protect from light.

Background / Technical Information:	Autophagy is a stress-induced protective mechanism. Less active under basal conditions, the mechanism is utilized by eukaryotic cells through lysosome-mediated bulk degradation of cellular contents when subjected to certain hostile conditions such as nutrient depletion and chemical or environmental stress. The role of increased autophagic activity in the pathology of cancer, neurodegeneration, cardiovascular disease and diabetes has become widely recognized and commonly studied. The Cyto-ID^® Autophagy Detection kit is a member of the CELLestial^® product line, reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications.

Product Literature References

Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4.: H.M. Warenius, et al.; Mol Cancer 10, 72 (2011), Abstract;

General Literature References

A live-cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment: J. Coleman, et al.; J. Biomol. Screen. 15, 398 (2010), Abstract;

Methods in mammalian autophagy research: N. Mizushima, et al.; Cell 140, 313 (2010), Abstract;

Assays to Assess Autophagy Induction and Fusion of Autophagic Vacuoles with a Degradative Compartment, Using Monodansylcadaverine (MDC) and DQ-BSA: C.L. Vazquez & M.I. Colombo; Methods Enzymol. 452, 85 (2009), Abstract;

Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux: M. Rossi, et al.; J. Cell Sci. 122, 3330 (2009), Abstract;

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes: D.J. Klionsky, et al.; Autophagy 4, 151 (2008), Abstract;