User Manual-ENZ-51010 - Rev 3.1 Feb 2012.pub
Contents
1. itself and gener ate 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 fluoro phore Check Methods and Procedures section of this manual and Appendix A for the recom mendations Minimal spectral overlap should occur with the selected set of filters 18 Life Sciences Enzo www enzolifesciences com Enabling Discovery in Life Science GLOBAL HEADQUARTERS EUROPE ASIA Enzo Life Sciences Inc Enzo Life Sciences ELS AG 10 Executive Boulevard Industriestrasse 17 Farmingdale NY 11735 CH 4415 Lausen Toll Free 1 800 942 0430 Switzerland Phone 631 694 7070 Phone 41 0 61 926 89 89 Fax 631 694 7501 Fax 41 0 61 926 89 79 info usa enzolifesciences com info ch enzolifesciences com For local distributors and detailed product information visit us online www enzolifesciences com2. perform a pretreatment by adding the ROS Inhibitor to the aliquots of ROS Superoxide Detection Mix for the last 30 minutes of the reagent loading Treatment with an experimental test agent or ROS inducer included with the kit should be performed in the cell culture media without dye Centrifuge the cells at 400 x g for 5 minutes 6 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 Immediately 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 NOTE Different exposure times may be required for optimal detec tion of the two dyes used in the kit E FLOW CYTOMETRY ADHERENT CELLS 1 The day before the experiment seed the cells on appropriate tis sue culture plates to ensure 50 70 confluency on the day of the experiment IMPORTANT Cells should be healthy and not overcrowded since results of the experiments will depend significantly on the cel
3. produced by peroxides peroxynitrite and hydroxyl radicals will be detected in the FL1 channel Superoxide production will be detected in the FL2 channel To avoid overlap between green and orange fluorescent signals the following compensation procedure should be performed 1 Run the unstained uninduced sample first Generate a FSC versus SSC dot plot and gate out cell debris 12 2 Generate a log FL1 X axis versus a log FL2 Y axis dot plot Adjust PMT voltages for both channels so the signals from unstained cells should fall within the first log decade scale of FL1 and FL2 axes Run single stained Green positive control and adjust FL2 FL1 compensation until the orange fluorescence signal will fall into the first decade of the log FL2 scale Repeat compensation procedure with the Orange single stained positive control and adjust FL1 FL2 compensation until the green fluorescence signal will fall into the first decade of the log FL1 scale NOTE It is important to use the brightest positive single stained samples for proper compensation correction that allows distin guishing between negative and slightly positive dim cells D ANTICIPATED RESULTS FLUOESCENCE MICROSCOPY 1 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 cytoplasm
4. visualized using a wide field fluorescence microscope equipped with standard green 490 525 nm and orange 550 620 nm filter set or cytometrically using any flow cytometer equipped with a blue 488 nm laser 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 microscopy assays or 50 flow cytometry assays using live cells adherent or in suspension Reagent Quantity Oxidative Stress Detection Reagent Green 300 nmoles Superoxide Detection Reagent Orange 300 nmoles ROS Inducer Pyocyanin 1 umole ROS Inhibitor N acetyl L cysteine 2x 10mg Wash Buffer Salts 1 pack Additional Materials Required e CO incubator 37 C e Standard fluorescence microscope or flow cytometer equipped with a blue laser 488 nm e Calibrated adjustable precision pipetters preferably with disposable plastic tips e 5 mL round bottom polystyrene tubes for holding cells during induction of ROS RNS for suspension cells only and during staining and assay procedure e Adjustable speed centrifuge with swinging buckets 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 u
5. 4 F FLUORESCENCE MICROPLATE ASSAY DATA ANALYSIS AND ANTICIPATED RESULTS 1 It is critical that positive pyocyanin induced and control untreated samples be included in every experiment for every cell type Negative NAC pretreated sample is optional but very helpful In preliminary experiments it is important to establish appropriate doses of inducers and inhibitors for each cell type used Cells with increased production of superoxide demonstrate bright orange fluorescence in the presence of the Superoxide Detection Reagent and the signal will be detected using standard rhodamine filter settings Cells with increased levels of oxidative stress demonstrate a bright green staining in the presence of the Oxidative Stress Detection Reagent and the signal will be detected using standard fluorescein filter setting ROS positive control samples induced with ROS Inducer Pyocyanin exhibit both bright orange and green fluorescence and can be detected in both green and orange channels Cells pretreated with the ROS Inhibitor N acetyl L cysteine should not demonstrate significant green or orange fluorescence upon induc tion Control untreated samples should present only low autofluores cence signal in both channels NOTE Remember that different cell types demonstrate different redox profiles Therefore the auto fluorescence signal may vary significantly in both channels Results of the experiments should be normalized usi
6. D WAG NRA J Enzo Enabling Discovery in Life Science og og o0 a a a mf oe odd odd odd EZE EE oor DUDU DUDU DUDU Cet es oe mn a a a Total ROS Superoxide Detection Kit for fluorescence microscopy flow cytometry and microplate assay Instruction Manual Cat No ENZ 51010 200 fluorescence microscopy assays or 50 flow cytometry assays or 2 x 96 well plates For research use only Rev 3 1 February 2012 Notice to Purchaser The Total ROS Superoxide 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 This product is of
7. ECTIONS ed ai daka Eih EA 12 B SETTING UP OPTIMAL EXPOSURE TIME FOR DETECTION OF THE DYES ege eer EEE 12 C COMPENSATION CORRECTION FOR FLOW CNTOMETRN 12 D ANTICIPATED RESULTS FLUORESCENCE MICROSCOPY 13 E FLOW CYTOMETRY DATA ANALYSIS AND ANTICIPATED RESULTS 14 F FLUORESCENCE MICROPLATE ASSAY DATA ANALYSIS AND ANTICIPATED RESULTS ssssisesiieeseesriiteriiteriintrinnerirtere esent 15 ReTCre NCS a sees iegegeg eebe Seege cede ee nnan 16 Troubleshooting Guide cccseeceesesseeeseeeeeeees 17 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 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 Total ROS Superoxide Detection Kit enables detection of comparative levels of total ROS RNS and also allows determi nation of superoxide production in live cells Through the co
8. de Detection Reagent Orange is supplied lyophilized and should be reconstituted in 60 uL anhydrous DMF to yield a 5 mM stock solution concentration Upon reconstitution the stock solution should be stored at 20 C Gently mix before use 2 Positive Control 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 recom mended 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 Control The ROS Inhibitor N acetyl L cysteine should be reconstituted in 123 uL of deionized water to yield a 0 5 M stock solution 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 being tested Endogenous fluorescence of untreated cells should be determined in advance or per assay 4 1X Wash Buffer Prepare 1X Wash Buffer by dissolving the contents of the pack in 1 liter of deionized water When not in use the 1X Wash Buffer should be stored refrigerated Warm to room temperature before use 5 ROS Superox
9. ed to avoid overlap between the green and orange fluorescent signals Recommended controls for flow cytometry e Unstained untreated cells e Positive control cells pyocyanin treated stained with Oxida tive Stress Detection Reagent Green only Green cells e Positive control cells pyocyanin treated stained with Super oxide Detection Reagent Orange only Orange cells G FLUORESCENCE MICROPLATE ASSAY ADHERENT CELLS 1 The day before the experiment seed the cells in 96 well black wall clear bottom plates at a density of 1 2 x10 cells per well to ensure 70 80 confluency on the day of the experiment Leave several wells empty for the background fluorescence control measurements IMPORTANT Cells should be healthy and not overcrowded since results of the experiments will depend significantly on the cells condition Treat the cells for highest quality data use at least 6 8 repli cates for each condition tested with the desired inducers It is highly recommended that doses and time of treatment are estab lished first Establish positive controls at least 6 8 replicates by treatment of the cells with pyocyanin see step A 2 page 4 for 30 min at 37 C Leave cells untreated or treated with vehicle only for the negative control at least 6 8 replicates see step A 3 page 4 IMPORTANT The dose of reagent and time of treatment are cell dependent and should be determined experimenta
10. ell Check the cell count to confirm proper cell density For suspension cells careful re moval of supernatant after washing steps is critical as cells may be dislodged and washed away 17 Problem Potential Cause Suggestion High fluorescent background 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 fluoro phore Check Methods and Procedures section of this manual and Appendix A for the recom mendations Minimal spectral overlap should occur with the selected set of filters Wash step is necessary Follow 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 No decrease in the fluorescent signal after using a specific inhibitor 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
11. fered under a limited warranty The product is 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 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 csssseeesessen 2 Safety Warnings and Precautions 0 0 3 Methods and Procedures cs cccesssssceeeseeeeeeees 3 A REAGENT PREPARATIONS sesieesieessiissrrtstirttriinstrrnttrnntnrnnnsinnnt 3 B CELL PREPARATIONS ssessssessiiestitsriessrirtesirtstienttrnstrnnterrnerrrnnt 5 C FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT CELLS 5 D FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CELLS 6 E FLOW CYTOMETRY ADHERENT CELLS ee 7 F FLOW CYTOMETRY GUSPENSIONCELLS eeren 8 G FLUORESCENCE MICROPLATE ASSAY ADHERENT CELLS 9 H FLUORESCENCE MICROPLATE ASSAY SUSPENSIONCELLS 10 Appendices ssssssssnnssnnnnennnnnnnnnnnnnnnnnnnnnnnn nnmnnn nnmnnn 12 A lt FILTER SET SEL
12. ic 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 Green ROS positive control samples induced with ROS Inducer Pyocyanin exhibit a bright orange fluorescence in the nucleus 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 Untreated samples should present only low autofluorescent back ground signal in any channel 13 E FLOw CYTOMETRY DATA ANALYSIS AND ANTICIPATED RESULTS 1 It is critical that positive pyocyanin induced and control untreated samples be included in every experiment for every cell type Negative NAC pretreated sample is optional but very helpful In preliminary experiments it is important to establish appropriate doses of inducers and inhibitors for each cell type used Cell debris should be gated out using FSC versus SSC dot plot Generate a log FL1 X axis versus a log FL2 Y axis dot plot and add quadrants to it Adjust quadrants so the majority of control cells 80 90 will fall into lower left quadrant Keep the same quadrant gate throughout the assay NOTE Remember that different cell types demonstrate d
13. ide Detection Mix Prepare the ROS Superoxide Detection Mix as follows To every 10 mL of 1X Wash Buffer see step 4 or culture medium add 2 uL Oxidative Stress Detection Reagent Green and 2 uL Super oxide Detection Reagent Orange Gently mix To prepare smaller volumes of ROS Superoxide Detection Mix intermediate1 10 dilution of both Green and Orange detection reagents in 1X Wash Buffer or culture medium is recommended NOTE To perform the compensation correction single stained samples should be used Make sure single component solutions Green and Orange are available 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 confluency 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 Superoxide Detection Mix see step A 5 page 4 using a volume sufficient to cover the cell monolayer and incubate under normal tissue culture conditions for 1 hour Caref
14. ifferent redox profiles therefore the number of the cells in the lower left quadrant may vary significantly between the cell lines Cells with increased production of superoxide demonstrate bright orange fluorescence and will be detected using the FL2 channel Such cells will appear in the two upper quadrants of a log FL1 X axis versus a log FL2 Y axis dot plot Cells with increased levels of oxidative stress demonstrate a bright green staining in the presence of the Oxidative Stress De tection Reagent and can be registered in FL1 channel Such cells will appear in the upper and lower right quadrants of a log FL1 X axis versus a log FL2 Y axis dot plot ROS positive control samples induced with ROS Inducer Pyocyanin exhibit both bright orange and green fluorescence and appear to be positive in FL1 and FL2 channels The increase of the cell population in the upper left upper right and lower right quadrants will be registered Cells pretreated with the ROS Inhibitor N acetyl L cysteine should not demonstrate significant green or orange fluorescence upon induction Control untreated samples should present only low auto fluorescent background signal in any channel thus falling into the lower left quadrant on an FL1 versus FL2 dot plot Results of the experiments can be presented as percentage of the cells with increased ROS production or as increase in the mean fluorescence of the induced samples versus control 1
15. l black wall plates Leave several wells empty for the background fluorescence control measure ments IMPORTANT Cells should be healthy and not overcrowded since results of the experiments will depend significantly on the cells condition Remove the medium by centrifugation of the plate and re suspend the cells in 100 ul fresh medium containing the desired inducer s For highest quality data use at least 6 8 replicates for each condition tested It is highly recommended that doses and time of treatment are established first Establish positive controls at least 6 8 replicates by treatment of the cells with pyocyanin see step A 2 page 4 for 30 min at 37 C Leave cells untreated or treated with vehicle only for the negative control at least 6 8 replicates See step A 3 on page 4 IMPORTANT The dose of reagent and time of treatment are cell dependent and should be determined experimentally for each cell line being tested preliminary experimentation After treatment remove the media containing the inducers by cen trifugation of the plate Discard the supernatant Optionally the plates may be washed with 1X Wash Buffer see step A4 page 4 using 200 uL per well Discard the supernatant by centrifugation 10 Optional An additional set of plates may be pretreated with the inhibitor NAC see step A3 page 4 for 1 hour at 37 C After incubation remove the medium containing the inhibitor by centrifugatio
16. le with Rhodamine Ex Em 550 620nm NOTE Different exposure times may be required for optimal detection of the two dyes used in the kit D FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CELLS 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 centrifuged 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 Superoxide Detec tion Mix see step A 5 page 5 and incubate under normal tissue culture conditions for 1 hour with periodic shaking Centrifuge the cells at 400 x g for 5 minutes to remove the ROS Superoxide 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 ROS Inducer Pyocyanin Negative Control samples should be established by treatment with the ROS Inhibitor N acetyl L cysteine NOTE Cells should be treated with the ROS Inhibitor 30 minutes prior to induction All treatments should be performed under normal tissue culture conditions It is recommended to
17. 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 pyocyanin treated cells and adjust the exposure time to record a bright fluorescent 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 6 single color images for each dye for each sample C COMPENSATION CORRECTION FOR FLOW CYTOMETRY Signals
18. lls e Positive control cells pyocyanin treated stained with Super oxide Detection Reagent Orange only Orange cells F FLOW CYTOMETRY SUSPENSION CELLS 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 centrifuged at 400 x g for 5 minutes yielding a working cell count of 1 5 x 10 cells sample Induce the cells with an experimental test agent A separate positive control sample should be treated with the ROS Inducer Pyocyanin A negative control sample should be established by treatment with the ROS Inhibitor N acetyl L cysteine NOTE Cells should be pre treated with the ROS Inhibitor 30 minutes prior to induction All treatments should be performed under normal tissue culture conditions Centrifuge the cells at 400 x g for 5 minutes Discard supernata tant 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 500 uL of the ROS Superoxide Detection Mix see step A 5 page 5 and incubate 30 min at 37 C in the dark No washing is required prior to the flow cytometry analysis IMPORTANT Compensation correction should be perform
19. lls are in the log growth phase Band pass filters are too narrow or not optimal for fluorescent probes fluorescence microscopy Multiple band pass filters sets provide less light than single band pass ones Use correct filter for each fluorophore Check Methods and Procedures section of this manual and Appendix A for recom mendations Insufficient fluorescent dye concentration Follow the procedures provided in this manual Insufficient inducer concen tration Determine an appropriate concen tration 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 Orange signal may disappear over time because of subsequent reac tions of superoxide with other spe cies like NO Green signal may quench if con centration of product becomes 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 Overcompensation of the signal flow cytometry Change the values of compensa tion correction using single stained positive samples Follow recom mendation in the Appendix Cell density is too low in microplate w
20. lly for each cell line being tested preliminary experimentation Optional An additional set of plates may be pretreated with the inhibitor NAC see step A3 page 4 for 1 hour at 37 C Then treat NAC inhibited cells with the desired inducer and with pyo cyanin see step A 2 page 4 for 30 min at 37 C for positive control Leave a set of cells untreated as a negative control Refer to step 2 above After treatment remove the media containing the inducers Optionally the plates may be washed with 1X Wash Buffer using 200 uL per well see step A4 page 4 Add 100 uL well of ROS Superoxide Detection Mix see step A5 page 4 and incubate for 60 min at 37 C H Read the plates bottom reading without removing the detection mix using a fluorescence microplate reader and standard fluorescein Ex 488nm Em 520nm and rhodamine Ex 550nm Em 610nm filter sets If required Z factor may be calculated for each detection profile using the following formula zZ 1 3 SDsampiet 3 S D controt Meansample Meancontroil FLUORESCENCE MICROPLATE ASSAY SUSPENSION CELLS NOTE Perform all steps requiring centrifugation at 400 x g for 5 minutes using a centrifuge with swinging buckets 1 Collect the cells by centrifugation at 400 x g for 5 min re suspend them in the appropriate cell culture medium at a density of 0 5 x 10 1 0 x 10 cells mL count and aliquot 100 uL of the suspension into wells of 96 wel
21. ls condition Induce the cells with an experimental test agent Separate posi tive control sample should be treated with the ROS Inducer Pyocyanin Negative Control samples should be established by treatment with the ROS Inhibitor N acetyl L cysteine NOTE Cells should be pre treated with the ROS Inhibitor at least 30 minutes prior to induction All treatments should be performed under normal tissue culture conditions Remove the media with the inducers inhibitors from the cells by aspiration Carefully wash cells twice with 1X Wash Buffer in a volume sufficient to cover the cell monolayer aspirate the super natant Detach cells from the tissue culture plates using any appropriate method collect cells in 5 mL round bottom polystyrene tubes and wash them with 1X Wash Buffer Centrifuge the cell suspension for 5 min at 400 x g at room temperature Discard the super natant 5 Resuspend the cell pellet in 500 uL of ROS Superoxide Detec tion Mix see step A 5 page 5 Stain cells for 30 min at 37 C in the dark No washing is required prior to the analysis of the samples by flow cytometry IMPORTANT Compensation correction will be needed to avoid over lap between green and orange fluorescent signals see Appendix C Recommended controls for compensation correction e Unstained untreated cells e Positive control cells pyocyanin treated stained with Oxida tive Stress Detection Reagent Green only Green ce
22. mbination of two specific fluorescent probes the kit provides a simple and specific assay for the real time measurement of global levels of reactive oxygen species ROS peroxynitrite 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 and or flow cytometry The kit includes two fluorescent dye reagents as major components Oxidative Stress Detection Reagent Green for ROS detection and Superoxide Detection Reagent Orange The non fluorescent cell permeable ROS detection dye green probe reacts directly with a wide range of reactive species such as hydrogen peroxide H202 peroxynitrite ONOO hydroxyl radicals Oe nitric oxide NO and peroxy radical ROOs yielding a green fluorescent product indicative of cellular production of different ROS RNS types However the green probe has a low sensitivity for superoxide Oz as well as Hypochlorous acid HCIO and nitric oxide NO relative to other free radicals The superoxide detection dye orange probe is a cell permeable probe that reacts specifically with superoxide ze 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 two dyes can be
23. n of the plate Discard the supernatant Then re suspend the NAC inhibited cells in 100 uL medium containing the desired inducer s and pyocyanin for positive control see step A 2 page 4 for 30 min at 37 C Leave a set of un induced NAC treated cells as a negative control Refer to step H 2 on page 10 After treatment remove the media containing the inducers by centrifugation of the plate Discard the supernatant Optionally the plates may be washed with 1X Wash Buffer see step A4 page 4 using 200 uL per well Discard the supernatant by centrifugation Re suspend the cells with 100 uL well of ROS Superoxide Detec tion Mix see step A5 page 4 and incubate for 60 min at 37 C Read the plates without removing the detection mix using a fluo rescence microplate reader and standard fluorescein Ex 488nm Em 520nm and rhodamine Ex 550nm Em 610nm filter sets If required Z factor may be calculated for each detection profile using the following formula Z 1 a 3 SDsampie 3 S D controt Mean sample em Mean gclll 11 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 other transmitted However it is important to realize that dichroic filters have a some what
24. ng the background readings from empty wells see step H1 and can be presented as ratios of the mean fluorescence of the induced samples versus control for each channel 15 4 02 4 N 2 T E 10 80 2 yey ane 3 17 10 10 10 10 UN FL1 C g 51 4 19 7 By 123 9 38 2 10 ef ae p x EE eer ER 10 10 10 19 10 FL1 D uae 25 N SES iw wer Vil Figure 1 Jurkat cells were induced with 100 uM pyocyanin general ROS inducer panel B 200 uM antimycin A superoxide inducer panel C or 1 uM of tbutyl hydroperoxide peroxide inducer panel D stained with two color ROS Detection Kit and analyzed using flow cytometry Untreated cells panel A were used as a Cell debris were ungated and compensation was performed using single stained pyocyanin treated samples Red numbers reflect the percentage of the cells control in each quadrant References a FY bh Tarpey M and Fridovich 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 Zhang J H et al J Bio Screening A 1999 VIII Troubleshooting Guide Problem Potential Cause Suggestion Low or no fluores cent signal in posi tive control Dead or stressed overcrowded cells Prepare fresh cell culture for the experiments Make sure that the ce
25. se only and is not intended for diagnostic purposes 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 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 thereagents 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 1 Detection Reagents 1 1 The Oxidative Stress Detection Reagent Green is supplied lyophilized and should be reconstituted in 60 uL anhydrous DMF to yield a 5 mM stock solution Upon reconstitution the stock solution should be stored at 20 C Gently mix before use 1 2 The Superoxi
26. ully remove the ROS Superoxide 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 ROS Inducer Pyocyanin Negative Control samples should be established by treatment with the ROS Inhibitor N acetyl L cysteine NOTE Cells should be treated with the 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 ROS Inhibitor to the aliquots of ROS Superoxide Detection Mix for the last 30 minutes of the reagent loading Treatment with an experimental test agent or ROS inducer included with the kit should be performed in the cell culture media without dye Carefully wash cells twice with 1X Wash Buffer in a volume sufficient to cover the cell monolayer Immediately overlay the cells with a cover slip and observe them under a fluorescence confocal microscope using standard excita tion emission filter sets Make sure 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 compatib
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