User Manual-ENZ-51001-200 - Rev 2.0 January 2010.pub
Contents
1. Insufficient inducer concen tration Determine an appropriate con centration of inducer for the cell line s used in the study Species of interest may react with each other thus attenuating the expected signal Check signaling pathways and all the components present in the cellular environment Inappropriate time point of the detection Make sure that time of detection is optimized and the samples are prepared immediately Red signal may disappear over time because the NO oxidized fluorescent probe can undergo further redox changes in the cellular physiological environ ment Green signal may quench if concentration of product be comes too high due to long exposure to the inducer Otherwise oxidized product may eventually leak out of the cells when left for a prolonged period 10 Problem Potential Cause Suggestion High fluorescent background No decrease of the fluorescent signal after using a specific inhibitor Stressed overcrowded cells Prepare new cell culture for the experiment Make sure that the cells are in the log growth phase Band pass filters are too narrow or not optimal for fluorescent probes Use correct filter for each fluorophore Check Methods and Procedures section of this manual and Appendix A for the recommendations Minimal spectral overlap should occur with the selected set of filters Wash step is necessary Follow2. 15 20 minutes upon treatment with NO Inducer L Arginine and may drastically decrease or disappear after that time Plan accordingly The ROS Inducer Pyocyanin is supplied lyophilized and should be reconstituted in 100 uL anhydrous DMF to yield a 10 mM stock solution For use a final concentration of 200 500 uM is recommended However the optimal final concentration is cell dependent and should be determined experimentally for each cell line being tested ROS induction generally occurs within 20 30 minutes upon pyocyanin treatment and may decrease or disappear after that time Plan accordingly 3 Negative Controls 3 1 3 2 3 3 The NO Scavenger c PTIO is supplied lyophilized and should be reconstituted in 100 uL DMF to produce a stock solution of 4 mM Foir use a final concentration of 20 80 uM is recommended However the optimal final concentration is cell dependent and should be determined experimentally for each cell line being tested Note Adherent cells pretreated with c PTIO may become weakly adherent and or detach from the cell culture substra tum The ROS Inhibitor N acetyl L cysteine should be reconsti tuted in 170 uL of deionized water to yield a 0 5 M stock solu tion N acetyl cysteine is not readily soluble and may require vortexing For use a final concentration of 5 mM is recommended However the optimal final concentration is cell dependent and should be determined experimentally for each cell line
3. 6 single color images for each dye for each sample C ANTICIPATED RESULTS 1 In the presence of NO the NO Detection Reagent Red will dem onstrate a red punctuate cytoplasmic staining pattern The Superoxide Detection Reagent Orange yields an evenly distributed bright orange nuclear staining pattern in induced cells Note the structural change in positively treated cells versus control untreated cells diffuse dim cytoplasmic structural pattern observed in the control cells is replaced with uniform cytoplasmic staining and bright nuclear staining in superoxide positive cells Increased levels of oxidative stress give a uniform green cytoplas mic staining in the presence of the Oxidative Stress Detection Reagent Nitric oxide NO positive control samples induced with NO Inducer L Arginine should exhibit red fluorescence with punctu ate cytoplasmic staining pattern Keep in mind that fluorescence of the NO Detection Reagent Red is not very bright Depending on the filters used the spill over of the red signal may be visible in the orange channel However in this case the staining pattern will differ significantly from the superoxide generated signal punctuate cytoplasmic staining versus nuclear staining More over this staining should disappear upon pretreatment with NO Scavenger c PTIO ROS positive control samples induced with ROS Inducer Pyocyanin exhibit a bright orange fluorescence in the nucl
4. Once produced within a cell free radicals can damage a wide variety of cellular constituents including proteins lipids and DNA However at lower concentrations these very same agents may serve as second messengers in cellular signaling Information rich methods are required to quantify the relative levels of various reactive species in living cells and tissues due to the seminal role they play in physiology and pathophysiology The ROS RNS Detection Kit enables detection of comparative levels of ROS RNS in cells and also distinguishes between different reactive species in live cells Through the combination of three specific fluorescent probes the kit provides a simple and specific assay for the real time measurement of free nitric oxide NO and by extension nitric oxide synthase NOS activity as well as global levels of reactive oxygen species ROS and specifically superoxide in living cells This kit is designed to directly monitor real time reactive oxygen and or nitrogen species ROS RNS production in live cells using fluorescence microscopy The kit includes three fluorescent dye reagents as major components NO Detection Reagent red fluorescent Oxidative Stress Detection Reagent green fluorescent for total ROS detection reagent and Superoxide Detection Reagent orange fluorescent The non fluorescent cell permeable NO detection dye reacts with NO in the presence of O2 with high specificity sensitivity and accuracy yielding a w
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6. as the inducer Note Cells should be treated with the NO Scavenger or ROS Inhibitor 30 minutes prior to induction All treatments should be performed under normal tissue culture conditions It is recommended to perform a pretreatment by add ing the NO Scavenger or ROS Inhibitor to the aliquots of ROS RNS 3 Plex Detection Mix for the last 30 minutes of the reagent loading Treatment with an experimental test agent or ROS NO inducers included with the kit should be performed in the cell cul ture media without dyes Carefully wash cells twice with 1X Wash Buffer in a volume sufficient to cover the cell monolayer Note Adherent cells pre treated with NO Scavenger c PTIO may become loose and or detach from the cell culture substratum Overlay the cells with a cover slip and observe them under a fluo rescence confocal microscope using standard excitation emission filter sets Make sure prepared samples are protected from dry ing Dried out cells may present different fluorescence patterns Recommended filter sets e Oxidative stress detection requires a filter set compatible with Fluorescein Ex Em 490 525nm e Superoxide detection requires a filter set compatible with Rhodamine Ex Em 550 620nm e NO detection requires a filter set compatible with Cyanine 5 650 670nm Note Different exposure times may be required for optimal detec tion of the three dyes used in the kit D FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CE
7. being tested Endogenous fluorescence of untreated cells should be determined in advance or per assay 4 1X Wash Buffer Allow the 10X Wash Buffer to warm to room temperature Make sure that the reagent is free of any crystallization before dilution Prepare enough 1X Wash Buffer for the number of samples to be washed by diluting each milliliter mL of the 10X Wash Buffer with 9 mL of deionized water 5 ROS RNS 3 Plex Detection Mix 5 1 Pre dilute the reconstituted Oxidative Stress Detection Reagent Green stock 1 10 using prewarmed tissue culture media 5 2 Pre dilute the reconstituted Superoxide Detection Reagent Orange stock 1 10 using prewarmed tissue culture media 5 3 Prepare the ROS RNS 3 Plex Detection Mix by combining appropriate volumes of the pre diluted Oxidative Stress De tection Reagent step 5 1 pre diluted Superoxide Detection Reagent step 5 2 and NO Detection Reagent Red using the volumes specified in the following table Note The NO Detection Reagent Red is ready to use and does not require a pre dilution step Important Prewarmed media should be used to avoid pre cipitation of the NO Detection Reagent Red in the mix ROS RNS 3 Plex Detection Mix Vol Req d per 1 ml Reagent Detection Mix NO Detection Reagent Red 2 5 uL Diluted 1 10 Oxidative Stress Detection 2 uL Reagent Green from step 5 1 Diluted 1 10 Superoxide Detection 2 uL Reagent Orange from step 5 2 Tissue C
8. gt D IZEARA OOOOO0000000c00c0 DDDDDDDDDDDDDDDDDD OOOGd00000000000 Enzo Enabling Discovery in Life Science ROS RNS Detection Kit for fluorescence microscopy Instruction Manual Cat No ENZ 51001 200 200 assays For research use only Rev 2 0 January 2010 Notice to Purchaser The ROS RNS 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 CELLestial reagents and kits are optimal for use in demanding imaging applications such as confocal microscopy flow cytometry and HCS 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 o
9. other means Methods and Procedures NOTE Allow all reagents to warm to room temperature before starting with the procedures Upon thawing of solutions 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 PREPARATIONS Reconstitution or dilution of any and all reagents in DMSO should be avoided as this solvent inhibits hydroxyl radical generation in cells Endogenous fluorescence of untreated cells should be determined in advance or per assay 1 Detection Reagents 1 1 The Oxidative Stress Detection Reagent Green is supplied lyophilized and should be reconstituted in 50 uL DMF to yield a 5 mM stock solution concentration Upon reconstitution stock solution should be stored at 20 C 1 2 The Superoxide Detection Reagent Orange is supplied lyophilized and should be reconstituted in 50 uL DMF to yield a 5 mM stock solution concentration Upon reconstitution stock solution should be stored at 20 C 2 Positive Controls 2 1 2 2 The NO Inducer L Arginine is supplied at a stock concentra tion of 100 mM in deionized water A final concentration of 1 mM is recommended However the optimal final concen tration is cell dependent and should be determined experi mentally for each cell line being tested NO induction generally occurs within
10. other transmitted However it is important to realize that dichroic filters have a some what limited reflectance range i e a 600 nm short pass dichroic filter may actually reflect light lt 500 nm When selecting filters it is critical to discuss with the filter or microscope manufacturer exactly what wavelength specifications are required for both the transmitted and the reflected light In addition filters should be obtained that have the highest possible transmission efficiency typically requiring anti reflection coating Each optic that an emission beam must traverse will remove some fraction of the desired light The difference between 80 transmis sion and 95 transmission for each detector may result in a greater than three fold difference in the amount of light available to the detec tor B SETTING UP OPTIMAL EXPOSURE TIME FOR DETECTION OF THE DYES Optimal exposure times should be established experimentally for each dye used in the experiment Both negative and positive controls should be utilized Start with the negative control untreated stained cells and set up the exposure time so the fluorescent background is negligible Then switch to a positive control arginine or pyocyanin treated cells and adjust the exposure time to record a bright fluores cent image Avoid saturation of the signal very bright spots on the image If saturation of the signal occurs decrease the exposure time It is recommended to acquire 5
11. the procedures provided in this manual making optional wash steps mandatory Inappropriate time point for detection Make sure that time of detection is optimized and the samples are prepared immediately Inappropriate cell conditions Make sure that you have viable cells at the beginning of the experiment and that the inducer treatment does not kill the cells during the time frame of the experiment Inappropriate inhibitor concentration too low or too high Very low doses of inhibitor may not affect ROS production by inducer Alternatively very high doses of the inhibitors may cause oxidative stress itself and generate fluorescent signal Optimize the concentration of the inhibitor and pretreatment time for each particular cell line Inappropriate time point for detection Inappropriate filter set on the microscope When cells are kept too long with the inhibitors or at very high inducer concentrations after a certain time the inhibitor becomes insufficient Make sure that time of detection is optimized Use correct filter for each fluorophore Check Methods and Procedures section of this manual and Appendix A for the recommendations Minimal spectral overlap should occur with the selected set of filters 11 www enzolifesciences com Enabling Discovery in Life Science Enzo Life Sciences North South America ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler
12. LLS 1 Cells should be cultured to a density not to exceed 1 x 10 cells mL Make sure 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 centri fuged at 400 x g for 5 minutes yielding a working cell count of 1 x 10 cells sample Resuspend the cell pellet in 200 uL of ROS RNS 3 Plex Detec tion Mix see page 5 and incubate under normal tissue culture conditions for 2 hours with periodic shaking Centrifuge the cells at 400 x g for 5 minutes to remove the ROS RNS 3 Plex Detection Mix Optional Resuspend the cells in 5 mL 1X Wash Buffer centrifuge them at 400 x g for 5 minutes and remove the supernatant Treat the cells with an experimental test agent Separate positive control samples should be treated with the NO Inducer L Arginine and the ROS Inducer Pyocyanin Negative control samples should be established by treatment with the ROS Inhibi tor N acetyl L cysteine or NO Scavenger c PTIO correspondingly for the same length of time as the inducer Note Cells should be treated with the NO Scavenger or ROS Inhibitor 30 minutes prior to induction All treatments should be performed under normal tissue culture conditions It is recommended to perform a pretreatment by adding the NO Scavenger or ROS Inhibitor to
13. ater insoluble red fluorescent product The non fluorescent cell permeable total ROS detec tion dye reacts directly with a wide range of reactive species such as hydrogen peroxide peroxynitrite and hydroxyl radicals yielding a green fluorescent product indicative of cellular production of different ROS RNS types The superoxide detection dye is a cell permeable probe that reacts specifically with superoxide generating an orange fluorescent product The kit is not designed to detect reactive chlorine or bromine species as the fluorescent probes included are relatively insensitive to these analytes Upon staining the fluorescent products generated by the three dyes can be visualized using a wide field fluorescence microscope equipped with standard green 490 525 nm orange 550 620 nm and red 650 670 nm fluorescent cubes Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored at 20 C or 80 C for long term storage 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 at least 200 assays using either live adherent cells or cells in suspension Reagent Quantity NO Detection Reagent Red 60 uL Oxidative Stress Detection Reagent Green 250 nmoles Superoxide Detection Reagent Orange 250 nmoles NO Inducer L Arginine 100 uL ROS Inducer Pyocyanin 1 umole NO Scave
14. eus as well as a bright green fluorescence in the cytoplasm Cells pretreated with the ROS Inhibitor N acetyl L cysteine should not demonstrate any green or orange fluorescence upon induction Cells incubated with the NO Scavenger c PTIO post induced with NO Inducer L Arginine should not demonstrate red fluores cence Untreated samples should present only low autofluorescent back ground signal in any channel Vil References 1 2 Tarpey M and Fridovich I Circ Res 89 2001 224 236 Batandier C etal J Cell Mol Med 6 2002 175 187 Gomes A ef al J Biochem Biophys Meth 65 2005 45 80 Wardman P Free Rad Biol Med 43 2007 995 1022 Vil Troubleshooting Guide Problem Potential Cause Suggestion Low or no fluorescent signal in positive control Dead or stressed overcrowded cells Prepare fresh cell culture for the experiments Make sure that the cells are in the log growth phase Band pass filters are too narrow or not optimal for fluorescent probes Multiple band pass filters sets provide less light than single band pass ones Use correct filter for each fluorophore Check Methods and Proce dures section of this manual and Appendix A for recommen dations Use of a wide band pass or a long pass filters is recommended in particular for the NO detection Insufficient fluorescent dye concentration Follow the procedures provided in this manual
15. f the purchase price All claims must be made to Enzo Life Sciences Inc within five 5 days of receipt of order Trademarks and Patents Enzo and CELLestial 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 Vi Vil VIII IptrodueHeont Age nieni enina EnA ea n 1 Reagents Provided and Storage sscssseeeseees 2 Additional Materials Required csssseeeseeeee 2 Safety Warnings and Precautions 0 3 Methods and Procedures ccccssececcessteeeeenseeeeeenes 3 A REAGENT PREDARATIONS 3 B CELL PREPARATIONS issssessessnsssessrenssssssrrrrsnssrsrrnnen 5 C FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT CELL 6 D FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CELL 7 Appendices ccceeeeeceeeseeeeeenseeeeeenseeneeenseeeeeenseeeenens 8 A FILTER SET GELECTION 8 B SETTING UP OPTIMAL EXPOSURE TIME FOR DETECTION OF THE DYES iiiiineeseeeeeeeseeerrnrseeerrne 8 C ANTIODATEDRESULUTES rene 9 e E 9 Troubleshooting Guide ccscesesesseeseeeeeees 10 Introduction Free radicals and other reactive species play influential roles in many human physiological and pathophysiological processes including cell signaling aging cancer atherosclerosis macular degeneration sepsis various neurodegenerative diseases Alzheimer s and Parkinson s disease and diabetes
16. nger c PTIO 400 nmoles ROS Inhibitor N acetyl L cysteine 2x 10mg 10X Wash Buffer 15 mL Additional Materials Required e CO incubator 37 C e Standard fluorescence microscope e Calibrated adjustable precision pipetters preferably with disposable plastic tips e Tubes appropriate for holding cells during induction of ROS RNS for suspension cells only e Adjustable speed centrifuge with swinging buckets for suspension cultures e Glass microscope slides e Glass cover slips e Deionized water e Anhydrous DMF 100 IV Safety Warnings and Precautions This product is for research use only and is not intended for diagnostic purposes The NO Detection Reagent Red contains DMF which is readily absorbed through the skin It is harmful if ingested or absorbed through the skin and may cause irritation to the eyes Observe appropriate precautions when handling 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 materials 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 environments or protected from light by
17. the aliquots of ROS RNS 3 Plex Detection Mix for the last 30 minutes of the reagent loading Treatment with an experimental test agent or ROS NO inducers included with the kit should be performed in the cell culture media without dyes 5 Centrifuge the cells at 400 x g for 5 minutes Resuspend the cells in 5 mL of 1X Wash Buffer centrifuge them at 400 x g for 5 minutes and remove the supernatant Resuspend the cells in 100 uL of 1X Wash Buffer and apply a 20 uL aliquot of the cell suspension sufficient for 2 x 10 cells onto a microscope slide Overlay the cells with a cover slip and analyze immediately via fluorescence microscopy Make sure that prepared samples are protected from drying Dried out cells may present different fluorescence patterns Recommended filter sets e Oxidative stress detection requires a filter set compatible with Fluorescein Ex Em 490 525nm e Superoxide detection requires a filter set compatible with Rhodamine Ex Em 550 620nm e NO detection requires a filter set compatible with Cyanine 5 650 670nm Note Different exposure times may be required for optimal detec tion of the three dyes used in the kit VI Appendices A FILTER SET SELECTION For fluorescence microscopy careful consideration must be paid to the selection of filters Dichroic filters should be selected in which the cut off frequency is optimally mid way between the two emission bands that are desired one reflected the
18. ulture Medium 993 5 uL Total Volume 1 mL Note Depending on the experiments dyes can be used separately according to a provided protocol B CELL PREPARATIONS Cells should be maintained via standard tissue culture practices Always make sure that cells are healthy and in the log phase of growth before using them for the experiment C FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT CELLS 1 The day before the experiment seed the cells directly onto glass slides or polystyrene tissue culture plates to ensure 50 70 con fluency on the day of the experiment Important Cells should be healthy and not overcrowded since results of the experiments will depend significantly on the cells condition Load the cells with the ROS RNS 3 Plex Detection Mix see page 5 using a volume sufficient to cover the cell monolayer and incubate under normal tissue culture conditions for 2 hours Carefully remove the ROS RNS 3 Plex Detection Mix from the glass slides by gently tapping them against layers of paper towel or from tissue culture plates Optional Cells may be washed with the 1X Wash Buffer Treat the cells with an experimental test agent Separate positive control samples should be treated with the NO Inducer L Arginine and the ROS Inducer Pyocyanin Negative control samples should be established by treatment with the ROS Inhibi tor N acetyl L cysteine or NO Scavenger c PTIO correspondingly for the same length of time
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