Cyto-ID™ Autophagy Detection Kit
Contents
1. Jo u Bunins i y oes ueiqui uu Buipuedx ue Aq si gy ABeydoyne uonoid p sioliqiuu se oid 45 Ly py upKuegdesq 5 ae spice ouluiy injonns Cu awososAjo ny _ 1 uuiosos 1 10 Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at lt 20 C protected from light When stored properly these reagents are stable for at least twelve months Avoid repeated freezing and thawing Reagents provided in the kit are sufficient for approximately 200 assays for flow cytometry application or 125 assays for fluorescence microscopy Reagent Quantity Cyto ID Green Detection Reagent 25 uL Hoechst 33342 Nuclear Stain 50 uL Autophagy Inducer Tamoxifen 50 mM 20 uL 10X Assay Buffer 30 mL Additional Materials Required Flow cytometer equipped with 488 nm laser source Standard fluorescence microscope Tubes appropriate for holding cells for the flow cytometer Calibrated adj2. Either increase the reagent concentration or increase the incubation time High Cyto ID Green dye staining observed in negative control sample Cell cultures overgrown Suspension cells should be cultured to a density not to exceed 1 x 10 cells and adherent cells should be approximately 50 70 confluent Growing cells in media for too long will cause depletion of nutrients Change media 4 8 hours before the experiment Pathogen infection Mycoplasm etc Obtain fresh cultures from reputable cell repository Cyto ID Green dye stained cells are too low to be readily quantified Cell density is either too low or cells are lost during process Increase cell density and gently aspirate supernatant during wash steps Cyto ID Green dye staining fails to stain in fixed and or permeabilized cells The dye is only suitable for live cell staining Use the dye only for live cell analysis Precipitate is observed in the 10X Assay Buffer Precipitate forms at low temperatures Allow solution to warm to room temperature or 37 C then vortex to dissolve all precipitate Cells do not appear healthy by microscopic examination Some cells require serum to remain healthy Add serum to the detection reagent and wash solutions Serum improves staining Typical amounts of serum to add range from 2 to 10 Tamoxifen treated cells ap pear dead or are no longe
3. LC3 protein Transfected Hela cells expressing RFP LC3 are treated with 10 uM Tamoxifen overnight Panel A Cyto ID Green staining Panel B RFP LC3 Panel C Composite images 01 0340 yey 5 unf Z9d H unf Zod H 9 199 81 81 81 81 81 81 81 9 81 81 81 81 81 9 say 0 9 07 001 OT 0 01 07 6 9 000 08 000 01 0 Y N sliqiuul sayeanoy sayeanoy sayeanoy Aseydoyne syquyuy ASeydoyne sajyeanoy ASeydoyne syqiyuy Aseydoyne syiqiyuy ASeydoyne sayeanoy ASeydoyne sayeanoy ASeydoyne sayeanoy ASeydoyne sayeanoy ASeydoyne sayeanoy 1999 Hd 05 7 59212 10 Iqiuu 9An39 9S 1u pu d pul u01ui 5 s np 3s uo8e 10 1 5 229 2110 03 0 s 9np 1 289 Hd jewososh 592 1 sjang 289 1 0 0 s 9n
4. cytometry Results are presented by histogram overlays Control cells were stained as well but mostly display low fluorescence In the samples treated with 10 uM Tamoxifen for 18 hours Cyto ID Green dye signal increases about 2 fold indicating that Tamoxifen causes an increase in autophagy in Jurkat cells References 1 Vazquez and Colombo 2009 Assays to Assess Autophagy Induction and Fusion of Autophagic Vacuoles with a Degradative Compartment Using Monodansylcadaverine MDC and DQ BSA Methods in Enzy mology Volume 452 85 95 Klionsky et 2008 Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes Autophagy 4 2 151 175 Mizushima Yoshimori and Levine 2010 Methods in Mammalian Auto phagy Research Cell 140 313 326 Coleman et al 2010 A live cell fluorescence microplate assay suitable for monitoring vacuolation arising from drug or toxic agent treatment J Biomol Screen 15 4 398 405 Rossi et al 2009 Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux J Cell Sci 122 3330 3339 11 VIII Troubleshooting Guide Problem Potential Cause Suggestion Low Cyto IDTM Green dye staining in all treatments including positive control A low concentration of the Cyto ID Detection Reagent was used or the reagent was incubated with the cells for an insufficient length of time
5. that unlike the lysomotrophic dyes LysoTracker Red and Acridine Orange which primarily detect lysosomes the Cyto ID Green autophagy dye only weakly stains lysosomes while serving both as a selective marker of autolysosomes and earlier autophagic compartments This staining pat tern differs markedly from that achieved with Lyso ID Red dye as well which detects autophagosomes generated by chloroquine and bafilomy cin A treatment but not vacuoles associated with other stimuli such as serum starvation 1 Microscopy Under physiological conditions autophagy is a_ constitutive self degradative process involved both in basal turnover of cellular components and as an induced response to nutrient starvation in eukaryotes During autophagy portions of the cytoplasm are seques tered by elongation of double membrane structures called phagopho res which form vesicles called autophagosomes These vesicles then fuse with lysosomes to form autolysosomes where their contents are degraded by acidic lysosomal hydrolases for subsequent recycling Fig 1 page 2 A prominent mammalian protein known to specifically associates with the autophagosome membrane is LC3 ll When Cyto ID Green autophagy detection dye is incorporated into cells the accumulation of this fluorescent probe is typically observed in spherical vacuoles in the perinuclear region of the cell in foci dis Figure 3 Cyto ID Green dye mostly co localizes with RFP
6. BSA Absorbance and Fluorescence emission spectra 350 461nm for Hoechst 33342 panel B were determined in 1X Assay Buffer A FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION The selection of optimal filter sets for a fluorescence microscopy application requires matching the optical filter specifications to the spectral characteristics of the dyes employed in the analysis see Figure 2 Consult the microscope or filter set manufacturer for assis tance in selecting optimal filter sets for your microscope For flow cytometry fluorescence channel FL1 green or FL2 orange is recommended for analysis of the Cyto ID Green dye staining using a 488 nm laser source B EXPECTED RESULTS A number of methods have been devised to investigate the autophagy pathway and the steps involved in the maturation of autophagosomes to autolysosomes acid hydrolase rich organelles in which the sequestered cytoplasmic material is ultimately degraded For example cadaverine MDC has been determined to be a useful probe for the analysis of the autophagic process by fluorescence microscopy How ever this probe requires 365 nm UV illumination and thus is not compati ble with 488 nm excitation sources commonly implemented in flow cy tometry The Cyto ID Autophagy Detection Kit employs a 488 nm excitable green emitting fluorescent probe to highlight the various vacuo lar components of the autophagy pathway It should be noted
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8. Enz0 Enabling Discovery in Life Science Cyto IDTM Autophagy Detection Kit for flow cytometry and fluorescence microscopy Instruction Manual Cat No ENZ 51031 K200 For research use only Rev 1 3 April2011 Notice to 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 exten sively benchmarked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytometry microplate readers and HCS HTS where consistency and reproducibility are required This product is manufactured and sold by ENZO LIFE SCIENCES INC for research use only by the end user in the research market and is not intended for diagnostic or therapeutic use Purchase does not include any right or license to use develop or otherwise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authorization of ENZO LIFE SCIENCES INC is strictly prohibited Limited Warranty These products are offered under a limited warranty The products are guaranteed to meet appropriate specifications described in the package insert at the time of shipment Enzo Life Sciences sole obligation is to replace the product to the extent of the purch
9. PY SUSPENSION CELLS 1 Cells should be cultured to a density not to exceed 1 x 10 cells mL Ensure that cells are in the log phase of growth before starting an experiment IMPORTANT Cells should be healthy and not overcrowded since results of the experiments will depend significantly on the cells overall condition A sufficient volume of cells should be centrifuged at 400 x g for 5 minutes yielding a working cell count of 1 x 10 cells sample Centrifuge cells for 5 minutes at 400 x g at room temperature RT to obtain a cell pellet Carefully remove the supernatant by aspiration and dispense 100 uL of Dual Detection Reagent from step 4A 3a page 4 to cover the cell pellet Protect samples from light and incubate for 30 minutes at 37 C Optional Wash the cells with 100 uL 1X Assay Buffer Remove excess buffer Re suspend cells in 100 uL 1X Assay Buffer Apply the cell suspension to a glass microscope slide and overlay with a coverslip Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the autophagic signal Optionally image the nu cleus using a DAPI filter set E Live CELL ANALYSIS BY FLOW CYTOMETRY 1 Cells should be maintained via standard tissue culture practice Grow cells overnight to log phase in a humidified incubator at 37 C 5 IMPORTANT Cells should be healthy and not overcrowded sinc
10. agosome with the lysosome The agents generate a positive signal in the Cyto ID Autophagy detection assay Furthermore MG 132 a potent cell permeable and selective protea some inhibitor has been shown to induce autophagy as demonstrated with the described assay The ubiquitin proteasome system UPS and autophagy serve as two complementary reciprocally regulated protein degradation systems Blockade of UPS by MG 132 is well known to activate autophagy Flow Cytometry Figure 4 on page 11 shows the typical results of flow cytometry based analysis of cell populations using the Cyto ID Autophagy Detection kit Control uninduced and 10 uM Tamoxifen treated Jurkat T Cell leukemia were used After 18 hours treatment cells were loaded with Cyto ID Green Detection Reagent then analyzed without washing by flow cytometry Results are presented by histogram 10 overlays Control cells were stained as well but display low fluores cence In the samples treated with 10 uM Tamoxifen 18 hours The Cyto ID TM Green dye signal increases about 2 fold indicating that Tamoxifen causes an increase in autophagic vesicles in Jurkat cells VII Figure 4 Flow cytometry based profiling of Cyto ID Autophagy Detection Kit Control uninduced and 10 uM Tamoxifen treated Jurkat cells T Cell leukemia were used After 18 hours treatment cells were loaded with Cyto ID Green Detection Reagent then ana lyzed without washing by flow
11. ase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo CELLestial and Cyto ID are trademarks of Enzo Life Sciences Inc Several of Enzo s products and product applications are covered by US and foreign patents and patents pending Contents Introduction ll Reagents Provided and Storage Additional Materials Required IV Safety Warnings and Precautions V Methods and Procedures A B C D E VI Appendices A B VII References VIII Troubleshooting Guide REAGENT PREPARATION aa CELL PREPARATIONS a LIVE CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT CELLS LIVE CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSIONCELLS LIVE CELL ANALYSIS BY FLOW CYTOMETRY FLUORESCENCE CHANNEL SELECTION FOR DATA COLLECTION EXPECTED RESULTS Introduction When subjected to certain hostile conditions that threaten survival such as when extracellular nutrients are limiting eukaryotic cells employ a lysosome mediated intracellular bulk degradation pathway for digesting their own cellular contents by a process referred to as autophagy Various cytoplasmic consti tuents including organelles and long lived protei
12. e results of the experiments will depend significantly on the cells overall condition Treat cells with compound of interest and negative control cells with vehicle Prepare positive control cells by incubating with the diluted Autophagy Inducer Tamoxifen 5 20 uM see section V A1 page 4 for 18 hours under normal tissue culture conditions At the end of the treatment trypsinize adherent cells or collect cells suspension cells Samples may contain 1 x 10 to 1 x 10 cells per mL Centrifuge at 400 x g for 5 minutes to pellet the cells Re suspend in media 1X Assay Buffer or other buffer of choice and centrifuge as before 6 Resuspend each live cell sample in 0 5 mL of freshly diluted Cyto ID Green Detection Reagent see step A 3b page 5 Incubate for 30 minutes at room temperature or 37 C in the dark It is important to achieve a monodisperse cell suspension at this step by gently pipetting up and down repeatedly 7 Analyze the samples in the green FL1 or orange FL2 channel of a flow cytometer No washing is required prior to the flow cytometry analysis VI APPENDICES z u Absorbance Fluorescence emission 400 500 200 250 300 350 400 450 500 550 600 Wavelength nm Wavelength nm Figure 2 Excitation and fluorescence emission spectra 463 534nm for Cyto ID Green dye panel A Spectra were determined in 10 mM sodium acetate buffer pH 4 with 3 mg mL
13. fer with 9 mL of deionized water Cyto ID Green Detection Reagent For optimal staining the concentration of the Cyto ID Green dye will vary depending upon the application Suggestions are provided to use as guidelines though some modifications may be required depending upon the particular cell type employed in the application a For fluorescence microscopy prepare a sufficient amount of Dual Detection Reagent for the number of samples to be assayed as follows For every 1 mL of 1X Assay Buffer see preparation in step 2 above or cell culture medium add 2 uL of Cyto ID Green Detection Reagent and 1 uL of Hoechst 33342 Nuclear Stain NOTE The dyes may combined into staining solution or each may be used separately if desired b The Hoechst 33342 Nuclear Stain can be diluted further if its staining intensity is much stronger than that of the Cyto ID Green dye c Some cells require serum to remain healthy Add serum to the detection reagent and wash solutions Serum im proves staining Typical amounts of serum to add range from 2 to 10 d When staining BFP or CFP expressing cells the Hoechst 33342 Nuclear Stain should be omitted due to its spectral overlap with these fluorescent proteins b For flow cytometry for each sample to be stained dilute 1 pL Cyto ID Green Detection Reagent to a final volume of 4 mL with media or buffer of choice B CELL PREPARATIONS Positive control ce
14. ging process A conventional fluorescent probe monodansylcadaverine MDC has served as a useful fluorescent marker for lysosomal autophagic vacuoles However it is known to generate high background and weak fluorescent signal Enzo Life Sciences Cyto ID Autophagy Detection Kit has been optimized for detection of autophagy in live cells by fluorescence microscopy and flow cytometry The assay provides a rapid specific and quantitative approach for monitoring autophagic activity at the cellular level The 488 nm excitable green fluores cent detection reagent supplied in the Cyto ID Autophagy Detection Kit becomes brightly fluorescent in vesicles produced during autophagy and has been validated under a wide range of conditions known to modulate auto phagy pathways Tamoxifen a known inducer of autophagy is included as a positive control in the kit A nuclear counterstain is provided in the kit as well to highlight this organelle This live cell analysis kit provides a convenient approach for the analysis of the regulation of autophagy at the cellular level y u osje Jo y u si pue y Anu nb sqns y 1 no y ue
15. lls should be pretreated with the Autophagy Inducer Tamoxifen for 6 18 hours Response to Tamoxifen is time and concen tration dependent and may also vary significantly depending upon cell type and cell line Negative control cells should be treated with a vehicle DMSO media or other solvent used to reconstitute or dilute an inducer or inhibitor for an equal length of time under similar conditions LIVE CELL ANALYSIS BY FLUORESCENCE CONFOCAL Microscopy ADHERENT CELLS 1 Grow cells on 18 x 18 mm coverslips or tissue culture treated slides inside a Petri dish filled with the appropriate culture medium When the cells have reached 50 70 level of confluence carefully re move the medium IMPORTANT should be healthy and not overcrowded as results of the experiments will depend significantly on the cells condition Dispense 100 uL of Dual Detection Reagent see section A 3a page 4 to cover the monolayer cells Protect samples from light and incubate for 30 minutes at 37 C Carefully wash the cells with 100 uL of 1X Assay Buffer see section V A2 Remove excess buffer and place coverslip upside down on microscope slide Analyze the stained cells by wide field fluorescence or confocal microscopy 60X magnification recommended Use a standard FITC filter set for imaging the autophagic signal Optionally image the nuclear signal using a DAPI filter set D Live CELL ANALYSIS BY FLUORESCENCE CONFOCAL MICROSCO
16. ns are sequestered into double membraned autophagosomes which subsequently fuse with lysosomes where their contents are degraded Under physiological condi tions autophagy plays a variety of important roles including maintenance of the amino acid pool during starvation damaged protein and organelle turnover prevention of neurodegeneration tumor suppression cellular differ entiation clearance of intracellular microbes and regulation of innate and adaptive immunity Autophagy is considered a dynamic multi step process which can be regulated at several steps in both a positive and negative manner Autophagic activity is typically low under basal conditions but can be markedly upregulated both in cultured cells and intact organisms by a variety of physiological stimuli such as nutrient starvation hypoxia energy depletion endoplasmic reticulum stress elevated temperature high density growth conditions hormonal stimulation pharmacological agent treatment innate immune signaling and in diseases such as viral bacterial or parasitic infections as well as various protein aggregopathies e g Alzheimer s Huntington s and Parkinson s disease heart disease and acute pancreatitis Autophagy can suppressed in certain other diseases including particular types of cancers neurodegenerative disorders infectious diseases and inflammatory bowel disorders A reduction in autophagic function is also considered a characteristic of the a
17. p 1 2 YEld yaya ayy s usiloqe pue Jo A 1 s se 19ul Hd 05 7 Juapuadapul YyoOLW 5 9 9 5 5 SHAUL Jo jo Jase jo 19812 amp 191 198161 ulpiuolo piuie1 do ulnboioju5 xoip H asojeyauy juawjealy tributed throughout the cytoplasm both locations depending upon the cell type under investigation A population of the Cyto ID Green autophagy dye labeled vesicles co localizes with LC3 a spe cific autophagosome marker Figure 3 Transfected HeLa cells ex pressing RFP LC3 were treated with either vehicle or 10 uM moxifen overnight The cells were then stained with Cyto ID Green dye Tamoxifen induces an increase in Cyto ID Green dye fluores cence intensity in punctuate structures that co localize with RFP LC3 Besides Tamoxifen treatment there are several other approaches known to induce autophagy One of the mos
18. r attached to the plate surface The of Tamoxifen may differ with different cell lines Try lowering the dose of Tamoxifen 12 Enzo Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA T 1 800 942 0430 610 941 0430 610 941 9252 info usa enzolifesciences com SWITZERLAND amp EUROPE ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 061 926 89 89 41 0 61 926 89 79 E _ info ch enzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 0 7621 5500 526 Toll Free 0800 664 9518 F 49 0 7621 5500 527 E info de enzolifesciences com www enzolifesciences com BENELUX ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium T 32 0 3 466 04 20 F 32 0 3 466 04 29 E info be enzolifesciences com www enzolifesciences com incorporating NTERNA UK amp IRELAND ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 8NL UK T 0845 601 1488 UK customers T 44 0 1392 825900 from overseas F 44 0 1392 825910 E info uk enzolifesciences com www enzolifesciences com www enzolifesciences com FRANCE ENZO LIFE SCIENCES c o Covalab s a s 13 Avenue Albert
19. s according to the operator s manual pertaining to the instrument being used NOTE Allow all reagents to thaw at room temperature before starting with the procedures Upon thawing gently hand mix or vortex the reagents prior to use to ensure a homogenous solution Briefly centrifuge the vials at the time of first use as well as for all subsequent uses to gather the contents at the bottom of the tube A REAGENT PREPARATION 1 Positive Control Tamoxifen is drug that is widely used in the treatment of breast cancer It competes with estrogen for receptor binding and prevents the transcription of estrogen responsive genes which results reduced cell growth Tamoxifen is thought to stimulate autophagy by increasing intracellular levels of ceramide and abolishing the inhibitory effect of the class pathway Tamoxifen induced autophagy is characterized by the accumulation of autophagic vacuoles and the stimulation of autophagic flux The Tamoxifen included in the kit is supplied as a 50mM solution To use it as a positive control dilute the Tamoxifen to 5 20 into your culture medium The agent has been validated in HeLa HepG2 and Jurkat cells 1X Assay Buffer Allow the 10X Assay Buffer to warm to room temperature Make sure that the reagent is free of any crystallization before dilution Prepare enough 1X Assay Buffer for the number of samples to be assayed by diluting each milliliter mL of the 10X Assay Buf
20. t potent known physio logical inducers of autophagy is starvation Autophagy induction can be observed with the Cyto ID Green dye within 1 hour of serum removal in both the HepG2 and HeLa cell lines Another approach to activate autophagy is through the modulation of nutrient sensing signaling pathways A popular target is mTOR which is a potent suppressor of autophagy Rapamycin an inhibitor of mTOR and ATP competitive inhibitors of mTOR such as PP242 increase Cyto ID Green dye signal Table 1 page 9 Several mTOR independent autophagy activators have also been validated using the Cyto ID Autophagy Detection Kit Table 1 Lithium induces autophagy through inhibition of inositol monophos phatase an mTOR independent pathway Trehalose and small molecule enhancers of rapamycin SMERs also induce autophagy by mechanisms that are not well understood Two FDA approved compounds that induce autophagy in an mTOR independent manner Loperamide hydrochloride and Clonidine also substantially increase green fluorescent signal in the assay Bafilomycin A1 is a selective inhibitor of vacuolar V type ATPases which results in elevated lysosomal pH Chloroquine verapamil nor clomipramine and hydroxychloroquine are small molecule modulators that passively diffuse into the lysosome and become trapped upon protonation All these agents also cause an increase in lysosomal pH which inhibits lysosome function and blocks fusion of the auto ph
21. ustable precision pipetters preferably with disposable plastic tips Adjustable speed centrifuge with swinging buckets for suspension cultures Deionized water Anhydrous DMSO Total growth medium suitable for cell type Glass microscope slides Glass cover slips 18 x 18 mm Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The Cyto ID Green Detection Reagent and the Autophagy Inducer Tamoxifen contain DMSO which is readily absorbed through the skin DMSO is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appropriate precautions when handling these reagents Reagents should be treated as possible mutagens and should be handled with care and disposed of properly Observe good laboratory practices Gloves lab coat and protective eyewear should always be worn Never pipet by mouth Do not eat drink or smoke in the laboratory areas All blood components and biological materi als should be treated as potentially hazardous and handled as such They should be disposed of in accordance with established safety procedures To avoid photobleaching perform all manipulations in low light environ ments or protected from light by other means 3 V Methods and Procedures The procedures described in this manual assume that the user is familiar with the basic principles and practices of flow cytometry and is able to run sample
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