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CYTO-ID® Autophagy detection kit 2.0

A brighter and more photostable, no-transfection, quantitative assay for monitoring autophagy in live  cells
 
ENZ-KIT175-0050 50 tests 219.00 USD
 
ENZ-KIT175-0200 200 tests 459.00 USD
Do you need bulk/larger quantities?
 
  • No transfection assay eliminates need for transfection efficiency validation
  • Brighter, more photostable dye specifically stains autophagic vesicles
  • Negligible staining of lysosomes reduces background seen with other dyes
  • Rapidly quantifies autophagy in native heterogeneous cell populations
  • Facilitates high-throughput screening of activators and inhibitors of autophagy
CYTO-ID® Autophagy Detection Kit 2.0 measures autophagic vacuoles and monitors autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated 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. Careful selection of titratable functional moieties on the dye prevents its accumulation within lysosomes, but enables labeling of vacuoles associated with the autophagy pathway.
Autophagy pathway
Schematic depiction of autophagy. Cytosolic material is sequestered by an expanding membrane sac, the phagophore, resulting in the formation of a double-membrane vesicle, an autophagosome. The outer membrane of the autophagosome subsequently fuses with the lysosome, and the internal material is degraded in the autolysosome. Various regulators of autophagy are also depicted in the diagram.
ENZ-KIT175 Spectra
Absorbance and fluorescence emission spectra (499/548nm) for CYTO-ID® Green Detection Reagent 2 (panel A) were determined in PBS. Absorbance and fluorescence emission spectra (350/461nm) for Hoechst 33342 (panel B) were determined in 1X Assay Buffer.
ENZ-KIT175 Microscopy
HeLa cells were stained with CYTO-ID® Green Detection Reagent 2 after being cultured in (A) full media or (B) starvation media (EBSS) with 40µM Chloroquine for 4h. Cells starved in EBSS in the presence of Chloroquine showed very bright green fluorescent signals and punctate structures.
ENZ-KIT175 FC
Flow cytometry-based profiling of autophagy in Jurkat cells. Jurkat cells were untreated or treated with 0.5µM Rapamycin (RAP), 10µM Chloroquine (CLQ) or both for 20h. After staining with CYTO-ID® Green Detection Reagent 2 for 30min, cells were washed and analyzed by flow cytometry. Results are presented as histogram overlay. Cells treated with RAP + CLQ show an increase in fluorescence.
ENZ-KIT175 Autophagy Induction
Microplate-based profiling of autophagy in HepG2 cells. HepG2 cells were stained CYTO-ID® Green Detection Reagent 2 after being cultured for 20h in DMSO (control), 0.5 µM Rapamycin (Rap), 10µM Chloroquine (CLQ), or both 0.5µM Rap and 10µM CLQ. Cells were also stained with Hoechst 33342 for cell number normalization. Cells treated with both Rapamycin and Chloroquine had an increase in autophagy, as measured by the CYTO-ID® Green Detection Reagent 2.
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Autophagy pathway ENZ-KIT175 Spectra ENZ-KIT175 Microscopy ENZ-KIT175 FC ENZ-KIT175 Autophagy Induction

Product Specification

Applications:Flow Cytometry, Fluorescence microscopy, Fluorescent detection, HTS
 
Application Notes:The CYTO-ID® Autophagy detection kit 2.0 provides a rapid, specific, and quantitative approach for monitoring autophagy in live cells by fluorescence microscopy, flow cytometry, and microplate reader.
 
Quality Control:A sample from each lot of CYTO-ID® Autophagy detection kit 2.0 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.
 
Quantity:For -0200 size:
200 flow cytometry assays, 250 microscopy assays or 3 x 96-well microplate assays.

For -0050 size:
50 flow cytometry assays, 60 microscopy assays or 1 x 96-well microplate assays.
 
Use/Stability:With proper storage, the kit components are stable for one year from date of receipt.
 
Handling:Protect from light. Avoid freeze/thaw cycles.
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Kit/Set Contains:CYTO-ID® Green Detection Reagent 2
Hoechst 33342 Nuclear Stain
Autophagy Inducer (Rapamycin)
Chloroquine Control
10X Assay Buffer
 
Scientific Background: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. Induction of autophagic flux can be visualized by enhanced accumulation of autophagic vesicles if lysosomal function is inhibited, preventing removal of these vesicles.  
 
Technical Info/Product Notes:The CYTO-ID® Autophagy Detection kit 2.0 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.
 
Protocol:A detailed protocol for FC in primary BMDCs can be found on bioprotocol.org: Flow Cytometric Analysis of Autophagic Activity with CYTO-ID Staining in Primary Cells by M. Stankov, et al.
 

Product Literature References

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells: K. Pant, et al.; Sci. Rep. 6, 34496 (2016), Application(s): Autophagosome formation assay, HepG2 cells, Abstract; Full Text

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;

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