User Manual-ENZ-51030-K100 - eFluxx
Contents
1. Reagents provided in the kit are sufficient for 100 flow cytometry assays using live cells adherent or in suspension Reagent Quantity eFluxx ID Gold Detection Reagent clear cap 2 vials MDR1 Inhibitor Verapamil white cap 300 nmoles MRP Inhibitor MK 571 yellow cap 750 nmoles BCRP Inhibitor Novobiocin purple cap 1 5 umoles Propidium lodide red cap 500 uL Additional Materials Required e CO incubator 37 C tissue culture plasticware e Water bath or thermostated dry block for 37 C incubations e Standard flow cytometer equipped with a blue laser 488 nm e Calibrated adjustable precision pipetters preferably with disposable plastic tips e 5mL round bottom polystyrene tubes for holding cells during staining and assay procedure e Adjustable speed centrifuge with swinging buckets e Anhydrous DMSO e Complete growth medium without Phenol Red e g Dulbecco s Modified Eagle medium D MEM e PBS optional for washing procedures IV Safety Warnings and Precautions e This product is for research use only and is not intended for diagnostic purposes e Some components of this kit may contain hazardous substances Reagents can be harmful if ingested or absorbed through the skin and may cause irritation to the eyes They should be treated as possible mutagens should be handled with care and disposed of properly e Observe good laboratory practices Gloves lab coat and protective eyewear should2. Red cells Set a second gate R2 in the FSC FL3 window to exclude Pl positive cells from analysis Figure 1B To avoid errors origi nated from the spillover of orange fluorescence of the eFluxx ID Gold Detection Reagent set the R2 border as high as possible Display R2 gated events in the FL2 histogram plot format Propidum lodide a e Ol aeee EE 0 200 o em eo 1000 o 200 40 60 0 1000 10 Forward Scatter Forward Scatter 10 10 10 FiworlD Gold Figure 1 Flow cytometry measurements of the samples Setting the parameters Gate out the debris Panel A gate Pl negative events Panel B and set PMTs for the FL2 fluorescence channel Panel C 5 Run tube no 1 and adjust the PMT amplification for FL2 so that the peak of the histogram is located between the second and third decades on the FL2 histogram channel Figure 1C Save settings in a properly designated template file e g MDR settings We recommend the use of the same or similar settings whenever possible You may need to readjust slightly the PMT amplifications and or the gate locations after an initial test run D CALCULATION OF RESULTS 1 Calculate the mean FL2 fluorescence intensity MFI values for each triplicate set of measurements Fuori from tubes no 1 2 and 3 Furp from tubes no 4 5 and 6 Fgcrp from tubes no 7 8 and 9 Fo from tubes no 10 11 and 12 If differences between parallel sets of measurements are
3. 0 5 mL of ice cold complete indicator free medium or PBS containing dilute Propidium lodide 50 uL of the provided stock solution of Propidium lodide in 5 mL of medium or PBS These samples can be stored at 4 C for several hours C FLOW CYTOMETRY MEASUREMENT OF SAMPLES The cellular orange fluorescence signal of e Fluxx ID Gold Detection Reagent should be measured using a flow cytometer in all tubes in the living PI negative cell population with identical equipment set tings 1 Within the flow cytometry software set an FSC SSC dot plot an FSC FL3 dot plot and an FL2 histogram plot For better separa tion of the different cell populations using a log scale is recom mended for the fluorescence channels FL2 and FL3 Run unstained cells and adjust both forward and side scatter PMT amplifications to display all the cell subsets on the FSC SSC dot plot Set a gate R1 on the FSC SSC dot plot selecting the cell popu lation of interest but excluding cell debris Figure 1A Display the cells selected by the R1 gate in an FSC FL3 dot plot format IMPORTANT Compensation correction may be needed to avoid overlap between orange and red fluorescent signals see Appendix C RECOMMENDED CONTROLS FOR COMPENSATION CORRECTION e Unstained untreated cells e Inhibitor verapamil is recommended treated cells stained with eFluxx ID Gold Detection Reagent Orange cells e Untreated cells stained with Propidium lodide only
4. MRP Inhibitor Resuspend the MRP Inhibitor MK 571 yellow cap in 75 uL anhydrous DMSO Vortex gently or slowly rotate the tube to dissolve Store the solution at 20 C The reconstituted reagent is stable for at least 6 months when stored as recommended 4 50 mM Novobiocin BCRP Inhibitor Resuspend the BCRP Inhibitor Novobiocin purple cap in 30 uL anhydrous DMSO Vortex gently or slowly rotate the tube to dissolve Store the solution at 20 C The reconstituted reagent is stable for 6 months when stored as recommended 3 B ASSAY PROTOCOL 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 an experiment IMPORTANT Because membrane transport mediated by ABC transport ers is a complex process that is highly dependent on physiological condi tions of cell populations and intracellular ATP status it is important to use living cells in good condition ATP depletion will decrease the activity of the membrane transporters 1 Grow cell line of choice in the appropriate medium Select drugs may interfere with dye efflux Therefore the cells should be kept in drug free medium for at least one week Anti microbial agent may be included in the medium since they do not interfere with multi drug resistance proteins Medium should be replaced one day before the assay Adherent cells should be dislodged from the plates using stan
5. introducing bubbles and incubate all the tubes at 37 C for 5 min Table 2 Assay Reagent Volumes O Vol Dilute Tube Nos Ire t B a Suspension S 1 3 125 uL MDR1 Inhibitor 250 pL 125 pL 4 6 125 uL MRP Inhibitor 250 pL 125 uL 7 9 125 uL BCRP Inhibitor 250 pL 425 pL RN 125 uL Medium DMSO 250 pL 125 pL 19 250 pL Medium DMSO 250 uL Unstained Control Dilute the eFluxx ID Gold dye stock solution from step V A1 by combining 16 uL of the stock solution and 2 mL of pre warmed 37 C complete indicator free medium Mix well by vortexing the tube gently IMPORTANT Dilution of the eFluxx ID Gold dye stock solution should be done just prior to use as it is susceptible to hydrolysis in aqueous solution Begin the assay by adding 125 pL of the freshly diluted eFluxx ID Gold dye solution from step B 7 into each tube except the tube labeled for unstained cells Gently mix cell suspensions by pipetting avoid introducing bubbles and incubate all the tubes at 37 C for 30 min After 25 min of incubation 5 uL of the provided stock solution of Propidium lodide red cap can be added to each tube for cell viability monitoring Perform flow cytometry measurements immediately after reaction proceed to section C page 6 If immediate measurements are not possible or if the number of samples is over 30 stop the reaction by rapid centrifugation 1 min 200 x g Discard the supernatant and re suspend the cells in
6. 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 CELLestial and eFluxx 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 LG Jotroductlon geesde ee 1 II Reagents Provided and Storage 2 III Additional Materials Required 2 IV Safety Warnings and Precautions 3 V Methods and Procedures 3 A REAGENT PREPARATION s s siisisesesisiriisrsirestsieresrseretne 3 B ASSAYPROTOCOL in teint onhati iets 4 C FLOW CYTOMETRY MEASUREMENTS OF SAMPLES 6 D CALCULATION OF RESULTS ei etreeeesreresrsrseeene 7 VI Appendices i ila ai 8 A SPECTRAL CHARACTERISTICS OF eFLUxX ID GOLD DETECTION REAGENT sissseesisiieiseseetrirreersereene 8 B TECHNICAL Hrs 8 C COMPENSATION CORRECTION ii 9 BD PRESUETS cu G Stet ees Ai tolo I io ee iii eee if AL 9 VII References neri 11 VIII Troubleshooting Guide 12 Introduction Multidrug resistance relates to resistance of tumor cells to a whole range of chemotherapy drugs with different structures and cellular targets The phe
7. J ENZO Enabling Discovery in Life Science eFluxx ID Gold Multidrug Resistance Assay Kit for flow cytometry Instruction Manual Cat No ENZ 51030 K100 for 100 assays For research use only Rev 1 0 1 October 2011 Notice to Purchaser The eFluxx ID Gold Multidrug Resistance Assay 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 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 other wise exploit this product commercially Any commercial use development or exploitation of this product or development using this product without the express prior written authori zation 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 product to the extent
8. acellular esterases The resulting probe is a hydrophilic fluorescent dye that is trapped within the cell unless actively pumped out by an ABC transporter The fluorescence signal of the dye generated within the cells thus depends upon the activity of the ABC trans porters The cells with highly active transporters will demonstrate lower fluorescence because of the active efflux of the reagent from the cell Appli cation of specific inhibitors of the various ABC transporter proteins in cluded in the kit allows differentiation between the three common types of pumps The activity of a particular MDR transporter is defined by the differ ence between the amount of the dye accumulated in the presence and in the absence of the inhibitors respectively The flow cytometry assay is based on determining fluorescence intensities of the tested cells after a short in vitro incubation of cell suspension with the eFluxx ID Gold Detection Reagent in the presence or absence of spe cific ABC transporter inhibitors The results of the test can be quantified by calculating the MDR activity factor MAF values which allow comparison of multidrug resistance between different samples or cell lines Reagents Provided and Storage All reagents are shipped on dry ice Upon receipt the kit should be stored upright and protected from light at lt 20 C When stored properly these reagents are stable for one year upon receipt Avoid repeated freezing and thawing
9. al 1996 Methods to detect p glycoprotein associated multidrug resistance in patients tumours Consensus recommendations Cancer Res 56 3010 3020 Gupta RS 1988 Intrinsic multidrug resistance phenotype of chinese hamster rodent cells in comparison to human cells Biochem Biophys Res Commun 153 598 605 McCollum AK TenEyck CJ Stensgard B et al 2008 P glycoprotein mediated resistance to Hsp90 directed therapy is eclipsed by the heat shock response Cancer Res 68 7419 7427 Collington GK Hunter J Allen CN Simmons NL Hirst BH 1992 Polarized efflux of 2 7 bis 2 carboxyethyl 5 6 carboxyfluorescein from cultured epithelial cell monolayers Biochem Pharmacol 44 417 24 Hunter J Hirst BH Simmons NL 1991 Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell HCT 8 and T84 layers expressing P glycoprotein Br J Cancer 64 437 44 Roelofsen H Vos TA Schippers lJ et al 1997 Increased levels of the multidrug resistance protein in lateral membranes of proliferat ing hepatocyte derived cells Gastroenterology 112 511 21 Hammond CL Marchan R Krance SM and Ballatori N Glutathione Export during Apoptosis Requires Functional Multidrug Resistance Associated Proteins J Biol Chem 282 14337 14347 Bodey B Taylor CR Siegel SE and Kaiser HE 1995 Immunocy tochemical observation of multidrug resistance MDR p170 glycoprotein expression in human osteosarcoma cells The clinical signifi
10. 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 e To avoid photobleaching perform all manipulations in low light environments or protected from light by other means Methods and Procedures NOTE PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING Allow all reagents to thaw at room temperature before starting with the proce dures 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 e Fluxx ID Gold Dye Stock Solution As needed resuspend the contents of each vial of eFluxx ID Gold Detection Reagent clear cap in 60 uL anhydrous DMSO Vortex gently or slowly rotate the tube to dissolve Store the solu tion at 20 C The reconstituted reagent is stable for at least 6 months when stored as recommended 2 5 mM Verapamil MDR1 Inhibitor Resuspend the MDR1 Inhibitor Verapamil white cap in 60 uL anhydrous DMSO Vortex gently or slowly rotate the tube to dissolve Store the solution at 20 C The reconstituted reagent is stable for at least 6 months when stored as recommended 3 10 mM MK 571
11. ative of a corresponding protein activity The numbers in the upper left corners are MAF scores multidrug resistance activity factors quantitative characteristics of multidrug resistance The eFluxx ID Gold Multidrug Resistance Assay Kit has been validated in various cell lines expressing multidrug resistance proteins that are summarized in Table 3 Table 3 Cell Lines Expressing Multidrug Resistance Proteins Tee son se son CHO K1 6 di HeLa 6 o S A549 7 e F HCT 8 8 9 S Hep G2 10 Jurkat 11 2 U 2 OS 12 U 2 OS RFP 12 Vil References 1 10 11 12 Homolya L Hollo Z Germann UA Pastan I Gottesman MM and Sarkadi B 1993 Fluorescent cellular indicators are extruded by the multidrug resistance protein J Biol Chem 268 21493 21496 Feller N Kuiper CM Lankelma J Ruhdal JK Scheper RJ Pinedo HM Broxterman HJ 1995 Functional detection of MDR1 P170 and MRP P190 mediated multidrug resistance in tumour cells by flow cytometry Br J Cancer 72 543 549 Broxterman HJ Lankelma J and Pinedo HM 1996 How to probe clinical tumour samples for P glycoprotein and multidrug resistance associated protein Eur J Cancer 32A 1024 1033 Litman T Druley TE Stein WD and Bates S 2001 From MDR to MXR new understanding of multidrug resistance systems their properties and clinical significance Cell Mol Life Sci 58 931 959 Beck WT Grogan TM Willman CL et
12. cance of MDR protein overexpression Anticancer Res 15 6B 2461 2468 11 VIII Troubleshooting Guide Problem Potential Cause Suggestion Cells do not exhibit fluorescence after incu bation with the detec tion reagent Cell viability is low Cells should be in log growth phase Cell samples can not be kept longer than 6 hours before the assay Do not use fixatives azides or other preservatives Avoid shear stress do not vortex and avoid bubbling Very low concentration of the eFluxx ID Gold Detection Reagent Check the concentration of the reagent Fluorescence does not increase after incuba tion with inhibitor Cells do not express any MDR1 MRP1 2 or BCRP Use a positive control cell type expressing corresponding ABC transporter Quality of the reagents is compromised Check storage stability and fresh ness of the reconstituted reagents Lower than expected fluorescence increases after incubation with inhibitor Overcompensation of the FL2 signal Change the values of compensa tion correction using single stained positive samples Follow the recommendations in Appendix C Percentage of dead PI positive cells is too high Wrong compensation correction Change the values of compensa tion correction using single stained positive samples Follow the recommendations in Appendix C There are differences in MFI mean fluores cence intens
13. dard methods and used in suspension for the assay Count cells using a hemacytometer For one sample test four assays should be performed in triplicates with three different inhibitors and without inhibitor Approximately 2 5 x 10 cells are required for each assay Prepare a cell suspension containing 1 2 x 10 cells mL in pre warmed 37 C complete indicator free medium For each sample to be assayed prepare four sets of tubes in triplicate Include one tube for the unstained cell control Immediately prior to use prepare intermediate dilutions of all 4 inhibitors by mixing appropriate volumes of inhibitor stock solu tions from step V A and pre warmed 37 C complete indicator free medium using the volumes specified in the following table IMPORTANT Prepare the dilutions of the inhibitors immediately prior to use as they are susceptible to hydrolysis in aqueous solution Add 125 uL of inhibitor into separate tubes as indicated in the following table For control add 125 uL pre warmed 37 C com plete indicator free medium containing 5 DMSO Table 1 Dilution of Inhibitors Inhibitor Vol Inhibitor Cone n Vol Medium MDRI1 Inhibitor white cap 16 uL 5 mM 1 mL MRP Inhibitor yellow cap 20 uL 10 mM 1 mL BCRP Inhibitor purple cap 8 uL 50 mM 1 mL 6 7 10 11 Add 250 uL of cell suspension into each tube see Table 2 Gen tly mix the contents of each tube by pipetting avoid
14. ity values between cell lines Detection reagent accumulation may be influenced by cell size endogenous esterase activity etc Using the inhibitors and calculating the MAF values eliminate these differences Inconsistent fluores cence shift using the same cells irreproducible results Inadequate incubation condition Always use a water bath not incu bator Ensure temperature of water bath is 37 C Cell viability is low Cells should be in log growth phase Cell samples cannot be kept longer than 6 hours before the assay Do not use fixatives azides or other preservatives Avoid shear stress do not vortex and avoid bubbling 12 Life Sciences www enzolifesciences com Enabling Discovery in Life Science NORTH SOUTH AMERICA ENZO LIFE SCIENCES INC 10 Executive Boulevard Farmingdale NY 11735 4710 USA T 1 800 942 0430 631 694 7070 F 631 694 7501 E _ info usa enzolifesciences com www enzolifesciences com EUROPE amp ASIA ENZO LIFE SCIENCES ELS AG Industriestrasse 17 Postfach CH 4415 Lausen Switzerland T 41 0619268989 F 41 0619268979 E info ch enzolifesciences com www enzolifesciences com GERMANY ENZO LIFE SCIENCES GMBH Marie Curie Strasse 8 DE 79539 L rrach Germany T 49 07621 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 Frankrijk
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16. lt 10 use all three values to calculate the mean If one value is extreme a difference of gt 10 disregard the unreliable data and calcu late the mean from the other two If all MFI values differ by gt 10 redo the analysis see the troubleshooting guide Calculate the multidrug resistance activity factor MAF for each transporter using the following formulas MAF wort 100 x Fori si Fo Fmpri MAF mrp 100 x Furr Fo Fmrp MAFgcrp 100 x Fgcrp Fo Fgcrp In extreme cases without MDR1 MRP or BCRP activity the MFI values corresponding to inhibitor treated cells can be smaller than the MFI value of non inhibitor treated cells In such cases corresponding MAF values should be regarded as zero VI APPENDICES A SPECTRAL CHARACTERISTICS OF eFLuxx ID GOLD DETECTION REAGENT 100 Figure 2 The absorption and emission peaks of e Fluxx ID Gold detection dye are 530 nm and 555 nm respectively It can be well excited with an argon ion laser at 488 wm wm did wm wm om om 700 nm and detected in the FL2 channel of Wavelength nm most bench flow cytometers B TECHNICAL HINTS 1 Multidrug resistance assay is a functional test that requires living cells in good condition The cells should always be kept in the appropriate incubation buffer containing all the essential compo nents Do not use fixatives azide or other preservatives Shear stress can be harmful to the cells Do not vortex cell
17. nomenon of multidrug resistance MDR is a well known problem in oncol ogy and thus needs profound consideration in cancer treatment One of the underlying molecular rationales for MDR is the up regulation of a family of transmembrane ATP binding cassette ABC transporter proteins that pre sent in practically all living organisms These proteins cause chemotherapy resistance in cancer by actively extruding a wide variety of therapeutic compounds from the malignant cells The same ABC transporters play an important protective function against toxic compounds in a variety of cells and tissues and at blood tissue barriers Enzo Life Sciences eFluxx ID Gold Multidrug Resistance Assay Kit is designed for functional detection and profiling of multidrug resistant pheno types in live cells both suspension and adherent The kit provides a fast sensitive and quantitative method for monitoring the function and expres sion of the three clinically most important multidrug resistance proteins MDR1 P glycoprotein MRP1 2 and BCRP The kit includes eFluxx ID Gold Detection Reagent as a major component Being a substrate for three main ABC transporter proteins this reagent serves as an indicator of these proteins activity in the cell The proprietary AM ester form of the eFluxx ID Gold Detection Reagent is a hydrophobic non fluorescent com pound that readily penetrates the cell membrane and is subsequently hy drolyzed inside of the cells by intr
18. scale Note It is important to use the brightest positive single stained samples inhibitor treated for proper compensation correction to allow negative cells to be distinguished from slightly positive dim cells D RESULTS 1 The theoretical range of the MAF values are between 0 and 100 Studies comparing MAF values with clinical response to a chemo therapeutic treatment suggest that a specimen with an MAF value of lt 20 can be regarded as multidrug resistance negative while MAF values gt 25 are indicative of multidrug resistance positive specimens In drug selected cell lines exhibiting extremely high expression levels of ABC transporter proteins the MAF values can be as high as 95 98 3 Typical results of the assay are presented in Figure 3 on page 10 MDR1 Inhibitor MRP Inhibitor BCRP Inhibitor 100 100 100 89 7 92 1 45 7 e so 0 3 Ze Ze 5 H 5 WI Wi WI 20 20 20 H D D 10 10 10 10 Ui 10 10 107 10 10 10 UV 10 10 mg eFluociD Gold eFluoID Gold eFluxx ID Gold FL2 Figure 3 Typical results of the multidrug resistance assay CHO K1 cells were incubated with eFluxx ID Gold Detection Reagent with and without specific inhibitors according to the kit protocol Resulting fluorescence was measured using flow cytometry Tinted histograms show fluorescence of inhibitor treated samples and non tinted histograms show fluorescence of untreated cells The difference in fluo rescence is indic
19. suspensions Always mix them with gentle pipetting Avoid form ing excessive bubbles Cells in suspension sediment very rapidly and have to be mixed prior to any procedure counting aliquotting running the samples on a flow cytometer Mix cells by gentle pipetting and forming excessive bubbles Cell suspensions at the recommended concentrations will nor mally result in 100 300 events sec flow rate Keeping the flow rate below 600 events sec is recommended C COMPENSATION CORRECTION Fluorescence of eFluxx ID Gold Detection reagent will be detected in the FL2 channel Dead non viable cells will be detected in the FL3 channel To avoid overlap between orange and red fluorescent signals the following compensation procedure should be performed 1 Run the unstained untreated sample first Generate a FSC versus SSC dot plot and gate out cell debris Generate a log FL2 X axis versus a log FL3 Y axis dot plot Adjust the PMT voltages for both channels so that the signals from the unstained cells falls within the first log decade scale of FL2 and FL3 axes Run single stained Red positive control and adjust the FL2 FL3 compensation until the orange fluorescence signal falls into the first decade of the log FL2 scale Repeat the compensation procedure with the Orange single stained positive control and adjust the FL3 FL2 compensation until the red fluorescence signal falls into the first decade of the log FL3
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