User Manual-ENZ-51012 - Rev 2.0 Jan 2010.pub
Contents
1. dark No washing is required prior to the flow cytometry analysis 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 limited reflectance range 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 DYE 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 up2. compared to a control cells fluorescence 5 ROS positive control samples induced with ROS Inducer Pyocyanin exhibit bright orange fluorescence and appear to be positive in FL2 channel 6 Cells pretreated with the ROS Inhibitor N acetyl L cysteine should not demonstrate significant green fluorescence upon induction 7 Control untreated samples should present only low autofluores cent background signal in any channel thus falling into the first decade on a log FL2 scale Results of the experiments can be presented as percentage of the cells with increased ROS production or as increase in the mean fluo rescence of the induced samples versus control Wile con 154 Wi con 154 ME pyo 203 ME ama 249 Figure 1 Jurkat cells were induced with 100 uM pyocyanin general ROS inducer panel A or 200 uM antimycin A superoxide inducer panel B stained with Superoxide Detection Reagent Orange and analyzed using flow cytometry Untreated cells tinted profile were used as a control Cell debris were ungated The numbers within the inserts reflect the mean orange fluorescence of the cells treated and control samples Vil References 1 Tarpey M and Fridovich Circ Res 89 2001 224 236 2 Batandier C et al J Cell Mol Med 6 2002 175 187 3 Gomes A et al J Biochem Biophys Meth 65 2005 45 80 4 Wardman P Free Rad Biol Med 43 2007 995 1022 VIII Troubleshooting Guide Pro
3. 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 VII VIII Introduction unsseisdaeteidandeutnddermeieee 1 Reagents Provided and Storage rrs rnnnnvnnnnnvnnnnvnnnn 1 Additional Materials Required runnnannvnnnnnnnnvnnnnnnnenn 2 Safety Warnings and Precautions rrrssvvrnnnnnvevnnnn 2 Methods and Procedures rrnssvrnnnvnnnnvnnnnnnnnnnnnnnnnvennn 3 A REAGENT PREPARATIONS csscsssesssssseessecssecsseeseeaseesseeseases 3 B CELL PREPARATIONS ccccscsssssssessseesseessecseessecssesseesseeasecsses 4 C FLUORESCENCE CONFOCAL MICROSCOPY ADHERENT CELLS urha inaina niian a a 4 D FLUORESCENCE CONFOCAL MICROSCOPY SUSPENSION CELLS escescsssesssscsseessecsseasecstecssecsceseesseesseess E FLOW CYTOMETRY ADHERENT CELLS F FLOW CYTOMETRY SUSPENSION CELLS ccsccsscesseesseeeees 6 Appendices rrnnnnvvnnnnnnvennnnnnvnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnvene 7 A FILTER SET SELECTION cccssscsssesssessscssecssecsscessessecasecsseeseeases 7 B SETTING UP OPTIMAL EXPOSURE TIME FOR DETECTION OF THE DYE u cssscsssssssesssesssessecsseesecsscasecssecsseess 7 C ANTICIPATED RESULTS FLUORESCENCE M
4. 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 7 Avoid saturation of the signal very bright spots on the image If saturation of the signal occurs decrease the exposure time It is rec ommended to acquire 5 6 single color images for each sample ANTICIPATED RESULTS FLUORESCENCE MICROSCOPY 1 It is critical that positive pyocyanin induced and control untreated samples be included in every experiment for every cell type Negative ROS Inhibitor pretreated sample is optional but very helpful In preliminary experiments it is important to estab lish appropriate doses of inducers and inhibitors for each cell type used 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 ROS positive control samples induced with ROS Inducer Pyocyanin exhibit a bright orange fluorescence in the nucleus Cells pretreated with the ROS Inhibitor N acetyl L cysteine should not demonstrate significant orange fluorescence upon induction Untreated samples should p
5. Enzo Enabling Discovery in Life Science Superoxide Detection Kit for fluorescence microscopy and flow cytometry Instruction Manual Cat No ENZ 51012 200 fluorescence microscopy assays or 50 flow cytometry assays For research use only Rev 2 0 January 2010 Notice to Purchaser The Superoxide Detection Kit is a member of the CELLestial product line reagents and assay kits comprising fluorescent molecular probes that have been extensively bench marked for live cell analysis applications CELLestial reagents and kits are optimal for use in demanding cell analysis applications involving confocal microscopy flow cytome try 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 offered 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
6. ICROSCOPY seiret eat 8 D FLOW CYTOMETRY DATA ANLYSIS AND ANTICIPATED RESULTS ccsccsssssssesseessessseesscesecssecaseesseesecases 8 References rrrnnnnnvnnnnnnnnnnnnnnnvnnnnnnnnnnnnnnnnnnnnnnnnnnnnnner 10 Troubleshooting Guide rrnnsevnnnnvnnnnvnnnnnvnnnnnnnnnner 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 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 Superoxide Detection Kit provides a simple and specific assay for the real time measurement of superoxide levels in living cells This kit is designed to directly monitor real time superoxide production in live cells using fluorescence microscopy and or flow cytometry A major component of the kit Superoxide Detection Reagent Orange is a cell permeable probe that reacts specifically with superoxide generating an ora
7. a cover slip and analyze via fluorescence microscopy Superoxide detection requires a filter set compatible with Rhodamine Ex Em 550 620nm Make sure that prepared samples are protected from drying Dried out cells may present different fluorescence patterns 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 cells 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 th
8. aining Solution as follows To every 10 mL of 1X Wash Buffer see step 4 or culture medium add 2 ul Superoxide Detection Reagent Orange Gently mix To prepare smaller volumes of Superoxide Staining Solution intermediate1 10 dilution of the Superoxide Detection Reagent Orange in 1X Wash Buffer or culture medium is recommended 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 Superoxide Staining Solution see step A 5 above using a volume sufficient to cover the cell monolayer and incubate under normal tissue culture conditions for 1 hour Carefully remove the Superoxide staining Solution 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 Cont
9. and assay procedure Adjustable speed centrifuge with swinging buckets Glass microscope slides Glass cover slips Deionized water Anhydrous DMF 100 Safety Warnings and Precautions This product is for research use only and is not intended for diagnos tic 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 V 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 Reagent The Superoxide Detection Reagent Ora
10. blem 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 cells 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 pro vide less light than single band pass ones Use correct filter set s Check Methods and Procedures section of this manual and Appen dix A for recommendations 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 such as NO 10 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 pas
11. e super natant Resuspend the cell pellet in 500 mL of Superoxide Staining Solution see step A 5 page 4 Stain cells for 30 min at 37 C in the dark No washing is required prior to the analysis of the sam ples by flow cytometry 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 2 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 3 Centrifuge the cells at 400 x g for 5 minutes Discard supernata tant 4 Resuspend the cells in 5 mL of 1X Wash Buffer centrifuge them at 400 x g for 5 minutes and remove the supernatant 5 Resuspend the cells in 500 uL of the Superoxide Staining Solu tion see step A 5 page 4 and incubate 30 min at 37 C in the
12. f this manual and Appendix A for the recom mendations 11 www enzolifesciences com Enabling Discovery in Life Science Enzo Life Sciences North South America ENZO LIFE SCIENCES INTERNATIONAL INC 5120 Butler Pike Plymouth Meeting PA 19462 1202 USA Tel 1 800 942 0430 610 941 0430 Fax 610 941 9252 info usa enzolifesciences com Switzerland amp Rest of Europe ENZO LIFE SCIENCES AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland Tel 41 061 926 89 89 Fax 41 061 926 8979 info ch enzolifesciences com Benelux ENZO LIFE SCIENCES BVBA Melkerijweg 3 BE 2240 Zandhoven Belgium Tel 32 0 3 466 04 20 Fax 32 0 3 466 04 29 info be enzolifesciences com Germany ENZO LIFE SCIENCES GmbH Marie Curie Strasse 8 DE 79539 Lorrach Germany Tel 49 0 7621 5500 526 Fax 49 0 7621 5500 527 into de enzolifesciences com incorporating UK amp Ireland ENZO LIFE SCIENCES UK LTD Palatine House Matford Court Exeter EX2 SNL UK Tel 0845 601 1488 UK customers Tel 44 0 1392 825900 overseas Fax 44 0 1392 825910 info uk enzolifesciences com For Local Distributors please visit our Website BIOCHEMICALS LEXIS BIOMOL
13. nge is supplied lyophi lized 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 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 Negative Control The ROS Inhibitor N acetyl L cysteine should be reconstituted in 170 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 How ever 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 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 Superoxide Staining Solution Prepare the Superoxide St
14. nge fluorescent product The kit is not designed to detect reactive peroxide hydroxyl peroxynitrite chlorine or bromine species as the fluo rescent probe included is relatively insensitive to these analytes Upon staining the fluorescent product generated can be visualized using a wide field fluorescence microscope equipped with standard orange e g 550 620 nm or red e g 650 670 nm fluorescent cubes 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 fluorescence microscopy assays or 50 flow cytometry assays using live cells adherent or in suspension Reagent Quantity Superoxide Detection Reagent Orange 300 nmoles ROS Inducer Pyocyanin 1 umole ROS Inhibitor N acetyl L cysteine 2x 10 mg Wash Buffer Salts 1 pack Additional Materials Required CO incubator 37 C Standard fluorescence microscope or flow cytometer equipped with a blue laser 488 nm Calibrated adjustable precision pipetters preferably with disposable plastic tips 5 mL round bottom polystyrene tubes for holding cells during induction of ROS RNS for suspension cells only and during staining
15. resent only low autofluorescent back ground signal in any channel 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 ROS Inhibitor pretreated sample is optional but very helpful In preliminary experiments it is important to estab lish 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 FL2 X axis versus FSC or SSC Y axis dot plot and add quadrants to it Adjust quadrants so the majority of con trol cells 80 90 will fall into lower left quadrant Keep the same quadrant gate throughout the assay Alternatively log FL2 histo gram can be used where the mean fluorescence of the peak for the untreated cells should fall within the first decade of a log FL2 scale NOTE Remember that different cell types demonstrate different redox profiles therefore the number of the cells in the lower left quadrant may vary significantly between the cell lines 4 Cells with increased production of superoxide demonstrate bright orange fluorescence and will be detected using FL2 channel Such cells will appear in the two right quadrants of a log FL2 X axis versus FSC or SSC dot plot If log FL2 histogram is used the peak generated by the superoxide positive cells will have increased FL2 fluorescence
16. rol 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 adding the ROS Inhibitor to the aliquots of Superoxide Staining Solution 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 compatible with Rhodamine Ex Em 550 620nm Make sure prepared samples are protected from drying Dried out cells may present different fluorescence patterns 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
17. s filters are too narrow or not optimal for fluorescent probes Use correct filter set Check Methods and Procedures section of this manual and Appendix A for the recommendations 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 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 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 Inappropriate filter set on the microscope Use correct filter for excitation and emission Check Methods and Procedures section o
18. sample Resuspend the cell pellet in 200 uL of Superoxide Staining So lution see step A 5 page 4 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 Super oxide Staining Solution 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 perform a pretreatment by adding the ROS Inhibitor to the aliquots of Superoxide Staining Solution 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 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
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