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Cyto-ID® Autophagy detection kit

A No-transfection, Quantitative Assay for Monitoring Autophagy in Live Cells
 
ENZ-51031-0050 50 tests 150.00 USD
 
ENZ-51031-K200 200 tests 412.00 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. Careful selection of titratable functional moieties on the dye prevents its accumulation within lysosomes, but enables labeling of vacuoles associated with the autophagy pathway.
MOA ENZ-51031 web image
ENZ-51031 Fig1 web image
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.
ENZ-51031 Fig2 Accum-Flux website
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.
ENZ-51031 Data-3 webimage
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.
ENZ-51031 Fig4 Cyto-IDGrn MDC web image
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.
Microplate Rapamycin
Figure 6: Overnight incubation of HepG2 cells with Rapamycin, an inhibitor of mTOR kinase, results in an increase in Cyto-ID® dye signal.
Please mouse over
MOA ENZ-51031 web image ENZ-51031 Fig1 web image ENZ-51031 Fig2 Accum-Flux website ENZ-51031 Data-3 webimage ENZ-51031 Fig4 Cyto-IDGrn MDC web image Microplate Rapamycin

Product Specification

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.
 
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.
 
Quantity:For -K200 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.
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Kit/Set Contains:Cyto-ID® Green Detection Reagent
Hoechst 33342 Nuclear Stain
Autophagy Inducer (Rapamycin)
Autophagy Inducer (Chloroquine)
10X Assay Buffer
 
Miscellaneous/General: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.
 
Background / Technical Information: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.

Application Note: Cell-Based Screening of Focused Bioactive Compound Libraries: Assessing Small Molecule Modulators of the Canonical Wnt Signaling and Autophagy-Lysosome Pathways.

Application Note: A Novel Image-Based Cytometry Method for Autophagy Detection in Living Cells.

Predictive Toxicology Application: Predictive High-Content/High-Throughput Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell (iPSC)-Derived Hepatocytes.
 
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

Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy of the protozoan parasite Trichomonas vaginalis: K. Y. Huang, et al.; Biochim. Biophys. Acta. 1840, 53 (2014), Abstract;
Autophagy in the brain of neonates following hypoxia-ischemia shows sex-and region-specific effects: S. N. Weis, et al.; Neuroscience 256, 201 (2014), Abstract;
Autophagy is activated in systemic lupus erythematosus and required for plasmablast development: A. J. Clarke, et al.; Ann. Rheum. Dis. Epub ahead of print, (2014), Abstract; Full Text
Cannabinoid-induced autophagy regulates suppressor of cytokine signaling-3 in intestinal epithelium: L.C. Koay, et al.; Am. J. Physiol. Gastrointest. Liver Physiol. 307, G140 (2014), Application(s): Detection of autophagy in human colonic epithelial cell line Caco-2 by Confocal imaging, Abstract; Full Text
Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium: T. Shiroto, et al.; PLoS One 9, 87871 (2014), Abstract; Full Text
Connective tissue diseases: How do autoreactive B cells survive in SLE-autophagy?: N. J. Bernard; Nat. Rev. Rheumatol. 10, 128 (2014), (Review), Abstract;
Defective Autophagosome Trafficking Contributes to Impaired Autophagic Flux in Coronary Arterial Myocytes Lacking CD38 Gene: Y. Zhang, et al.; Cardiovasc. Res. 102, 68 (2014), Abstract;
Defects in mitochondrial clearance predispose human monocytes to interleukin-1β hyper-secretion: R. van der Burgh, et al.; J. Biol. Chem. 289, 5000 (2014), Abstract; Full Text
Early biomarkers of response to carfilzomib in multiple myeloma (MM): Modulation of CXCR4 and induction of autophagy: M. Bhutani, et al.; J. Clin. Oncol. 32, e19572 (2014), Application(s): Quantification of autophagy in malignant plasma cells from bone marrow aspirates by flow cytometry with the Cyto-ID autophagy detection kit,
Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes: M. Xu, et al.; J. Cell. Mol. Med. (2014), Abstract; Full Text
Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells: M. Stankov, et al.; Bio-Protocol (2014), Application(s): FC in primary BMDCs, Full Text
High-Content Assays for Hepatotoxicity Using Induced Pluripotent Stem Cell-Derived Cells: O. Sirenko, et al.; Assay Drug Dev. Technol. 12, 43 (2014), Abstract; Full Text
Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy: M. V. Stankov, et al.; Leukemia 28, 577 (2014), Abstract;
Histone deacetylase inhibitors potentiate VSV oncolysis in prostate cancer cells by modulating NF-κB dependent autophagy: L. Shulak, et al.; J. Virol. 88, 2927 (2014), Abstract;
In vitro and in vivo characterization of porcine acellular dermal matrix for gingival augmentation procedures: A.M. Pabst, et al.; J. Periodontal. Res. 49, 371 (2014), Abstract;
Inhibition of Autophagic Flux by Salinomycin Results in Anti-Cancer Effect in Hepatocellular Carcinoma Cells: J. Klose, et al.; PLoS One 9, e95970 (2014), Application(s): Autophagy detection in human hepatocellular carcinoma , Abstract; Full Text
Inhibition of stress induced premature senescence in presenilin-1 mutated cells with water soluble Coenzyme Q10: D. Ma, et al.; Mitochondrion 17C, 106 (2014), Application(s): Autophagic vacuoles in Alzheimer's Disease fibroblasts detected with CytoID® Green Autophagy Detection kit, Abstract;
Involvement of autophagy in recombinant human arginase-induced cell apoptosis and growth inhibition of malignant melanoma cells: Z. Wang, et al.; Appl. Microbiol. Biotechnol. 98, 2485 (2014), Abstract;
MiR-216a: a link between endothelial dysfunction and autophagy: R. Menghini, et al.; Cell Death Dis. 5, 1029 (2014), Abstract;
Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells: E. Wolmarans, et al.; Cell. Mol. Biol. Lett. 19 , 98 (2014), Application(s): Autophagy detection in esophageal carcinoma SNO cell , Abstract;
Novel sorafenib-based structural analogues: in-vitro anticancer evaluation of t-MTUCB and t-AUCMB: A. T. Wecksler, et al.; Anticancer Drugs 25, 433 (2014), Abstract;
Photodynamic therapy with the novel photosensitizer chlorophyllin f induces apoptosis and autophagy in human bladder cancer cells: D. Lihuan, et al.; Lasers Surg. Med. 46, 319 (2014), Abstract;
Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells: Y.C.Li, et al.; Cancer Lett. 344, 239 (2014), Abstract;
Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer: D. Uchida, et al.; J. Gastroenterol Hepatol. 29, 973 (2014), Abstract;
Sirt1 modulates endoplasmic reticulum stress-induced autophagy in heart: A. Guilbert, et al.; Cardiovasc. Res. 103 (suppl 1), S13 (2014), Application(s): Evaluation of Autophagy in H9c2 cells, rat cardiomyoblasts by flow cytometry, Full Text
T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis: C.W. Lin, et al.; PLoS One 9, e102066 (2014), Application(s): Quantification of autophagosomes and autolysosomes staining in CD4+ and CD8+ cell population by flow cytometry , Abstract; Full Text
Tetracyclines cause cell stress-dependent ATF4 activation and mTOR inhibition: A. Brüning, et al.; Exp. Cell Res. 320, 281 (2014), Abstract;
The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells: K. Rothe, et al.; Blood 123, 3622 (2014), Application(s): Monitor autophagy flux in hematopoietic stem/progenitor cells, Abstract;
Androgen deprivation and androgen receptor competition by bicalutamide induce autophagy of hormone-resistant prostate cancer cells and confer resistance to apoptosis: B. Boutin, et al.; Prostate 73, 1090 (2013), Application(s): Measurement of autophagic flux in prostate cancer cells, Abstract;
Arenobufagin, a natural bufadienolide from toad vonem, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway: D.M. Zhang, et al.; Carcinogenesis 34, 1331 (2013), Application(s): Autophagy detection in hepatocellular carcinoma, Abstract;
Autophagy Plays a Critical Role in ChLym-1-Induced Cytotoxicity of Non-Hodgkin's Lymphoma Cells: J. Fan, et al.; PLoS One. 8, 72478 (2013), Abstract; Full Text
BCL-2 inhibitors sensitize therapy-resistant chronic lymphocytic leukemia cells to VSV oncolysis: S. Samuel, et al.; Mol. Ther. 21, 1413 (2013), Abstract;
Bleomycin exerts ambivalent antitumor immune effect by triggering both immunogenic cell death and proliferation of regulatory T cells: H. Bugaut, et al.; PLoS One 8, e65181 (2013), Application(s): Measurement of autophagy by flow cytometry and fluorescence microscopy, Abstract; Full Text
Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress: K. Suzuki, et al.; Neuro. Oncol. 15, 1186 (2013), Abstract;
Chloroquine Engages the Immune System to Eradicate Irradiated Breast Tumors in Mice: J. A. Ratikan, et al.; Int. J. Radiat. Oncol. Biol. Phys. 87, 761 (2013), Abstract;
Dietary Resveratrol Prevents Development of High-Grade Prostatic Intraepithelial Neoplastic Lesions: Involvement of SIRT1/S6K Axis: G. Li, et al.; Cancer Prev. Res 6, 27 (2013), Application(s): Effects of Resveratrol on prostate tumorigenesis, Abstract;
Enhancement of autophagy by simvastatin through inhibition of Rac1-mTOR signaling pathway in coronary arterial myocytes: Y.M. Wei, et al.; Cell. Physiol. Biochem. 31, 925 (2013), Abstract; Full Text
GX15-070 (obatoclax) induces apoptosis and inhibits cathepsin D and L mediated autophagosomal lysis in antiestrogen resistant breast cancer cells: J.L. Schwartz-Roberts, et al.; Mol. Cancer Ther. 12, 448 (2013), Application(s): Autophagy detection in breast cancer cells, Abstract;
Hydroxychloroquine preferentially induces apoptosis of CD45RO+ effector T cells by inhibiting autophagy: A possible mechanism for therapeutic modulation of T cells: J. van Loodregt, et al.; J. Allergy Clin. Immunol. 131, 1443 (2013), Application(s): Detection of autophagy in CD4+ T cells and PBMC by flow cytometry , Abstract; Full Text
Interactions between autophagic and endo-lysosomal markers in endothelial cells: C.L. Oeste, et al.; Histochem. Cell. Biol. 139, 659 (2013), Abstract;
Involvement of cholesterol depletion from lipid rafts in apoptosis induced by methyl-β-cyclodextrin: R. Onodera, et al.; Int. J. Pharm. 452, 116 (2013), Application(s): Measurement of autophagy by fluorescence microscopy, Abstract;
ISG15 deregulates autophagy in genotoxin-treated ataxia telangiectasia cells: S.D. Desai, et al.; J. Biol. Chem. 288, 2388 (2013), Application(s): Fluorescence microscopy using Ataxia Telangiectasia cells, Abstract; Full Text
Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells: Implications for glaucoma pathogenesis: K. Porter, et al.; Autophagy 9, (2013), Application(s): Autophagy detection by flow cytometry in porcine TM cells, Abstract; Full Text
Nelfinavir and bortezomib inhibit mTOR activity via ATF4-mediated sestrin-2 regulation: A. Brüning; Mol. Oncol. 7, 1012 (2013), Abstract;
Recombinant human arginase induced caspase-dependent apoptosis and autophagy in non-Hodgkin's lymphoma cells: X. Zeng, et al.; Cell Death Dis. 4, 840 (2013), Abstract; Full Text
Regulation of autophagic flux by dynein-mediated autophagosomes trafficking in mouse coronary arterial myocytes: M. Xu, et al.; Biochim. Biophys. Acta. 1833, 3228 (2013), Abstract;
Renal cancer-selective Englerin A induces multiple mechanisms of cell death and autophagy: R.T. Williams, et al.; J. Exp. Clin. Cancer Res. 32, 57 (2013), Application(s): Flow cytometry and immunofluorescence of a human kidney carcinoma cell line, Abstract; Full Text
Saxifragifolin D induces the interplay between apoptosis and autophagy in breast cancer cells through ROS-dependent endoplasmic reticulum stress: J.M. Shi, et al.; Biochem. Pharmacol. 85, 913 (2013), Application(s): Autophagy detection by flow cytometry in breast cancer cells, Abstract;
Suppression of autophagy enhanced growth inhibition and apoptosis of interferon-β in human glioma cells: Y. Li, et al.; Mol. Neurobiol. 47, 1000 (2013), Abstract;
Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage: A.V. Knizhnik, et al.; PLoS One 8, e55665 (2013), Application(s): Autophagy detection by flow cytometry in glioma cells, Abstract; Full Text
The effect of Zhangfei on the unfolded protein response and growth of cells derived from canine and human osteosarcomas: T. Bergeron, et al.; Vet. Comp. Oncol. 11, 140 (2013), Application(s): Detection of autophagy in human and canine osteosarcoma, Abstract;
The mTOR inhibitor RAD001 potentiates autophagic cell death induced by temozolomide in a glioblastoma cell line: E. Josset, et al.; Anticancer Res. 33, 1845 (2013), Abstract;
Therapeutic Combination of Nanoliposomal Safingol and Nanoliposomal Ceramide for Acute Myeloid Leukemia: T.J. Brown, et al.; J. Leuk. 1, 1000110 (2013), Application(s): Detection of autophagy by flow cytometry in Human HL-60 , HL-60/VCR, and murine C1498 cells, Full Text
Type I interferons induce autophagy in certain human cancer cell lines: H. Schmeisser, et al.; Autophagy 9, (2013), Application(s): Autophagy detection in type I interferon-treated human cancer cell lines, Abstract;
A novel image-based cytometry method for autophagy detection in living cells: L.L. Chan, et al.; Autophagy 8, 1371 (2012), Abstract; Full Text
Apoptosis and autophagy have opposite roles on imatinib-induced K562 leukemia cell senescence: C. Drullion, et al.; Cell Death Dis. 3, e373 (2012), Application(s): Flow cytometry of human CML cells treated with Imatinib, Abstract; Full Text
Counteracting autophagy overcomes resistance to everolimus in mantle cell lymphoma: L. Rosich, et al.; Clin. Cancer Res. 18, 5278 (2012), Abstract; Full Text
Heme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation of Apoptosis-and Autophagy-regulating Molecules: P. Banerjee, et al.; J. Biol. Chem. 287, 4962 (2012), Application(s): Detection of autophagy in human renal cancer cells, Abstract;
Inhibition of monocarboxylate transporter 2 induces senescence-associated mitochondrial dysfunction and suppresses progression of colorectal malignancies in vivo: I. Lee, et al.; Mol. Cancer Ther. 11, 2342 (2012), Abstract; Full Text
Mechanism for the induction of cell death in ONS-76 medulloblastoma cells by Zhangfei/CREB-ZF: T.W. Bodnarchuk, et al.; J. Neurooncol. 109, 485 (2012), Application(s): Detection of autophagy in medulloblastoma cells, Abstract;
Mitochondrial metabolism in Parkinson's disease impairs quality control autophagy by hampering microtubule-dependent traffic: D. M. Arduíno, et al.; Hum. Mol. Genet. 21, 4680 (2012), Abstract; Full Text
Proteasome inhibition by quercetin triggers macroautophagy and blocks mTor activity: A. K. Klappan, et al.; Histochem. Cell Biol. 137(1), 25 (2012), Abstract;
Reovirus as a viable therapeutic option for the treatment of multiple myeloma: C.M. Thirukkumaran, et al.; Clin. Cancer Res. 18, 4962 (2012), Application(s): Detection of autophagy in human myeloma cell lines and ex vivo tumor specimens, Abstract;
Src inhibition with saracatinib reverses fulvestrant resistance in ER-positive ovarian cancer models in vitro and in vivo: F.A. Simpkins, et al.; Clin. Cancer Res. 18, 5911 (2012), Application(s): Detection of autophagy in human ovarian cancer cells and xenografts, Abstract;
FoxM1 knockdown sensitizes human cancer cells to proteasome inhibitor-induced apoptosis but not to autophagy: B. Pandit, et al.; Cell Cycle 10, 3269 (2011), Application(s): Flow cytometry using human cancer cells, Abstract; Full Text
Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry: J.S. Lee, et al.; Methods 56(3), 375 (2011), Abstract;
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;
Silibinin triggers apoptotic signaling pathways and autophagic survival response in human colon adenocarcinoma cells and their derived metastatic cells: H. Kauntz, et al.; Apoptosis 16, 1042 (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;

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