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1. Lysate Preparation Considerations Before You Begin e Do not omit steps from the sample preparation protocol All steps are necessary for optimum assay performance USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 7 of 25 e fit is important to know the lysate protein concentration from cells grown in 96 well plates prepare additional wells of cells solely for this purpose e Ifusing cells grown in 96 well plates avoid plating cells in the outermost wells This minimizes cell growth edge effects Lysis Protocol for Cell Lines 1 Prepare 1X Assay Diluent by adding 25 ml 5X Assay Diluent WideScreen Reagent Kit to 100 ml sterile distilled deionized water Store 1X Assay Diluent that will be used within one month at 4 C To avoid microbial growth dispense working aliquots of any remaining 1X Assay Diluent and store at 20 C 2 Prepare 1X Wash Buffer by adding 20 ml 10X Wash Buffer WideScreen Reagent Kit to 180 ml sterile distilled deionized water Store at 4 C 3 Calculate the total amount of Extraction Reagent needed Prepare 10 excess to account for pipetting error Format Extraction Reagent T 175 flask 4 ml T 75 flask 2
2. 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 13 of 25 Note Notes 2 Calculate the protein concentration of the four fold diluted lysate samples based on the protein quantification values previously determined using BCA assay For example if the original sample concentration was 1 6 mg ml the dilution results in 400 ug ml If desired cell extracts can be further diluted to ensure more accurate signal quantification In this case follow the optional steps below Steps 3 5 within this section A range from 1 10 ug total cell protein per assay well is usually optimal 3 Label four centrifuge tubes In tube 1 mix the four fold diluted lysate and titration buffer to a final volume of 400 ul and final protein concentration of 100 ug ml 10 ug well in the assay For example if the four fold diluted extract has a total protein concentration of 400 pg ml mix 100 ul diluted extract with 300 pl titration buffer 4 Ifadditional dilutions of the extract are desired prepare three additional 2 fold dilutions of the cell extract as follows Add 150 ul titration buffer to tubes 2 3 and 4 Transfer 150 ul from tube 1 to the 150 ul titration buffer in tube 2 and mix well Change tips Transfer 150 ul from tube 2 to the 150 ul titration buffer in tube 3 Mix well Proceed in similar fashion with the serial dilutions through tube 4 5 These dilutions will result in 10 ug 5
3. Phycoerythrin PE Note Prepare 1X Streptavidin PE solution within 30 min of use 1 Briefly spin the tubes containing the 100X Streptavidin PE to collect reagent that might be trapped in the tube cap 2 Calculate the total volume of 1X Streptavidin PE solution required 100 ul is needed for each used well Note Do not dilute the whole vial of Streptavidin PE if the entire kit will not be used Dilute only what is needed based on the number of wells Allow 10 excess for pipetting error 3 Prepare the calculated volume of 1X Streptavidin PE solution by diluting Streptavidin PE Concentrate 1 100 in 1X Assay Diluent For example for 40 test wells 44 well including 10 extra add 44 pl Streptavidin PE to 4356 ul 1X Assay Diluent to prepare a final volume of 4400 ul Vortex 3 sec Protect from light and store at 4 C until use Wash wells three times with 1X Wash Buffer as described above After the final vacuum filtration tap filter plate on a paper towel to remove any buffer on the underside Immediately add 100 pl 1X Streptavidin PE solution to each well Incubate for 45 min at room temperature on a platform plate shaker 750 rpm Protect from light Optional Add 30 pl fixation solution to each well 0 2 paraformaldehyde in PBS not provided in the kit Incubate for 5 min at room temperature on a platform plate shaker 750 rpm Protect from light Note Fixation will improve well to well assay reproducibility 9 Wash wells three
4. and let it freeze 5 Add tissue and steel ball close the tube and pulverize for 1 minute at 2000 rpm 6 Incubate for 60 min on ice with occasional vortexing USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 9 of 25 7 Clarify lysate by centrifuging twice at 16 000 x g for 10 min at 4 C After each centrifugation transfer supernatants to new microcentrifuge tubes after each centrifugation step 8 Remove a 50 ul sample of each extract for protein quantification by BCA Protein Assay Cat No 71285 Determine the total protein concentration of each extract 9 Either proceed immediately to the Bead based Immunoassay Protocol on p 10 or store aliquots at 70 C Avoid multiple freeze thaw cycles Flowchart for Lysate Preparation Cells Grown in Flasks or plates Cell Lysis Discard culture medium Wash cells twice with ice cold TBS Add supplemented Extraction Reagent 4 ml per T 175 flask 2 ml per T 75 flask 1 ml per T 25 flask 200 ul per well of 6 well plate 120 ul per well of 96 well plate Incubate for 20 min on ice Filtration of cell lysates Pre wet filter or filter plate with TBS For lysates with volume gt 0 2 ml use a syringe tip filter p
5. ml T 25 flask 1 ml 6 well 200 ul well 96 well 120 ul well 4 Prepare the required volume of supplemented Extraction Reagent Per ml Extraction Reagent add 1 pl Protease Inhibitor Cocktail III 1000X 0 1 pl Benzonase Nuclease 20 ul Phosphatase Inhibitor Cocktail Set V 50X Notes Prepare fresh supplemented Extraction Reagent each time cell lysates are made Because the Breast Cancer Panel assays are not phospho specific the use of Phosphatase Inhibitor Cocktail Set V is optional 5 Aspirate and discard culture medium 6 On ice rinse cell monolayer twice with cold Tris buffered saline TBS Remove all TBS For non adherent cells transfer cells to centrifuge tubes centrifuge at 500 x g and wash twice with ice cold TBS 7 Add cold supplemented Extraction Reagent to adherent cells Incubate for 20 min at 4 C with gentle agitation rocking platform or occasional swirling For non adherent cells tap the centrifuge tube with finger to loosen cell pellet Add supplemented Extraction Reagent Incubate for 20 min at 4 C with occasional vortexing 8 Dislodge and solubilize all adherent cells using a rubber policeman or by repeated pipeting Extracts should be clear and non viscous 9 Clear lysates by filtration Pre wet filter or filter plate with TBS then remove all excess buffer For lysates with volume gt 0 2 ml use syringe tip filter pore size 0 45 um For lysates with volume lt 0 2 ml use a 96 we
6. pg 2 5 ug or 1 25 ug total cell protein per assay well respectively refer to figure below titration buffer TR aa 150 ul 150 ul 150 ul 150 wt 150 pN 150 pl 40 ug total cell protein to final volume of 400 ul with titration buffer i J J J J final total cell protein 10 ug 5 ug 2 5 ug 1 25 ug per well in xMAP assay Step 4 Prepare Capture Beads Prepare diluted Capture Beads within 1 h of use Individual Breast Cancer Panel Bead Kits can not be multiplexed together in all combinations because of incompatibilities between some assays Please refer to the compatibility chart in Appendix C on p 25 to determine optimal individual bead kit combinations 1 Calculate the number of test wells needed allowing 10 extra for pipetting error 2 Note the volume of 50X Capture Beads needed per well based on the assay format In all cases this results in approximately 3000 beads per bead region per well Assay Format Vol Capture Beads 50X needed Singleplex one target 1 ul per well User assembled multiplex 1 pl from each individual Bead Kit per well Breast Cancer Panel I II or III multiplex 1 ul per well premixed 3 Thoroughly resuspend each vial of Capture Beads 50X by vortexing for 10 sec sonicating in an ultrasonic bath for 10 sec optional and vortexing again for 5 sec USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom
7. see p 5 for storage conditions Note The Breast Cancer Panel Bead Kits and reagents are not necessarily compatible with other bead kits and reagents sold by Novagen or other vendors Please refer to Appendix C on p 25 for compatibility of assays found in WideScreen Breast Cancer Panels II and Ill USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 All Other Countries www novagen com novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 23 of 25 B Dilution Series for Generating Standard Curves The standard curve is used to quantify target proteins found in cell extracts and tissue samples Standards are recombinant fusion proteins representing all or part of the target proteins Notes Standard concentrations are assay dependent This is because the linear range and lower limit of each assay depends on assay sensitivity Values shown are the final concentrations in pg ml Standards supplied with Breast Cancer Panel Bead Kits for individual targets contain only the standard of interest but can be mixed with other standards see Appendix C for multiplex analysis Breast Cancer Panel I Standards M
8. times with 1X Wash Buffer as described above After the final vacuum filtration tap filter plate on a paper towel to remove any buffer on the underside 10 Immediately add 120 ul 1X Assay Diluent to the beads in each well To fully resuspend beads before running samples on the Luminex System incubate for 3 5 min on a platform plate shaker Protect from light 11 Analyze samples with a Luminex System according to the manufacturer s instructions USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 17 of 25 Flowchart for Breast Cancer Panel Immunoassay Prepare Titration Buffer 75 1X Assay Diluent 25 Extraction Reagent 2000 ul for duplicate standard curves 300 ul for each lysate sample diluted gt 4 fold final Prepare Capture Beads Prepare 7 point Standard Curve Prepare Sample Dilutions Vortex sonicate Capture Beads 50X 30 ul pre mixed Breast Cancer Panel Dilute lysate four fold in 1X Assay Per well Standards Mix in tube 1 or 30 ul Diluent 1 ul Capture Beads 50X from each bead each individual Breast Cancer Panel Prepare 100 ug ml dilution in kit Standard in tube 1 tube 1 10 ug lysate titration 1X Assay Diluent to final volume of 50 ul Bri
9. 56 ul 1980 ul Step 5 Combine Capture Beads with Analytes 1 On the 96 well filter plate tape off wells that are not going to be used for the assay with the provided Plate Sealer cut to size or lab tape for future use 2 Pre wet the 96 well filter plate for at least 5 min by adding 50 ul 1X Assay Diluent to each well that will be used With the vacuum manifold apply gentle vacuum 3 in 76 mm Hg to filter plate just until liquid aspiration is complete Do not let the vacuum exceed 5 in 127 mm Hg Tap filter plate on a paper towel to remove any buffer on the underside Notes It is critical to remove excess buffer from the underside of the filter plate before adding samples or reagents Otherwise samples may wick out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations See Considerations Before You Begin on p 10 for guidelines on using the filter plate vacuum and manifold If well does not drain use the non tip end of a fresh 200 pipet tip to flick the center of the plastic support on the underside of the well and then reapply the vacuum 3 Vortex 10 sec the diluted Capture Beads solution prepared as per Step 4 Prepare Capture Beads on p 13 Add 50 ul beads to each well being used 4 Remove liquid from filter plate by vacuum filtration To bead containing wells reserved for the standards add 100 ul from the standard dilutions Dilutions 1 7 bl
10. Store at 4 C 72085 3 HER 2 PAI 1 IGF 1R angiogenin Breast Cancer Panel II 5 Plex 100 tests EGER Fas VEGFR2 estrogen receptor a Store at 4 C 72086 3 TIMP 2 Breast Cancer Panel II 4 Plex 100 tests TIMP 1 uPA E Cadherin IGFBP 3 Store at 4 C 72087 3 Individual Breast Cancer Panel I Bead Kits 100 tests Angiopoietin 2 Total Bead Kit Store at 4 C 72075 3 100 tests Progesterone receptor Total Bead Kit Store at 4 C 72081 3 100 tests HER 2 Total Bead Kit Store at 4 C 71932 3 100 tests PAI 1 Total Bead Kit Store at 4 C 72080 3 100 tests IGF 1R Total Bead Kit Store at 4 C 71929 3 100 tests Angiogenin Total Bead Kit Store at 4 C 72074 3 Individual Breast Cancer Panel II Bead Kits 100 tests EGFR Total Bead Kit Store at 4 C 71928 3 100 tests Fas Total Bead Kit Store at 4 C 72078 3 100 tests VEGFR 2 Total Bead Kit Store at 4 C 71933 3 100 tests Estrogen receptor a Total Bead Kit Store at 4 C 72077 3 100 tests TIMP 2 Total Bead Kit Store at 4 C 72083 3 Individual Breast Cancer Panel III Bead Kits 100 tests TIMP 1 Total Bead Kit Store at 4 C 72082 3 100 tests uPA Total Bead Kit Store at 4 C 72084 3 100 tests E Cadherin Total Bead Kit Store at 4 C 72076 3 100 tests IGFBP 3 Total Bead Kit Store at 4 C 72079 3 USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Tol
11. TIMP 1 uPA E Cadherin IGFBP 3 Mol Wt kDa 20 55 87 29 Bead Region 23 26 15 19 pg ml pg ml pg ml pg ml Dilution 1 5000 30000 10000 3000 Dilution 2 1250 7500 2500 750 Dilution 3 313 1875 625 188 Dilution 4 78 469 156 47 Dilution 5 20 117 39 12 Dilution 6 5 29 10 3 Dilution 7 1 7 2 0 7 Blank 0 0 0 0 USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 25 of 25 C WideScreen Breast Cancer Panels Assay Compatibility Chart Some individual bead based assays should not be multiplexed together typically because detection antibodies from one assay will bind non specifically to capture beads from another assay This matrix indicates compatibility between all paired combinations of the WideScreen Breast Cancer Panel Bead Kits Not Compatible signifies a combination that should not be used together typically due to unacceptably high background in one of the assays Compatible with increased background combinations may be used with only a minor loss in sensitivity Compatibility with other bead based assays from Novagen or other vendors has not been tested Note This Compatibility Chart identifies cases of non specific interference between assays and is n
12. a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this product for a full refund prior to using it in any manner You the buyer acquire the right under Luminex Corporation s patent rights if any to use the product or any portion of this product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument The terms and conditions governing EMD Chemicals sale of this product are as indicated on our website www emdbiosciences com USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 3 of 25 About the Kits Cell Extraction Kit 1 kit 71926 3 WideScreen Reagent Kit 1 kit 71783 3 WideScreen Breast Cancer Panel II and III Kits See Appendix A Overview Bead based flow cytometric xMAP assays enable sensitive precise quantitation of analytes within a sample When directed toward proteins such assays are essentially sandwich immunoassays on a bead Samples are combined with fluorescently labeled microparticle
13. aS Novagen TM WideScreen Breast Cancer Panel I ll and Ill Table of Contents USA and Canada Tel 800 526 7319 novatech novagen com About E KITS siosana anea Ya lechs cc sebieh sal aa A ange oats 3 Overview 3 Components 4 Components and Storage 5 Additional Materials Required But Not Supplied 5 GrOWth OF Cell LINCS cesen a R 6 Considerations Before You Begin 6 Protocol for Growth of Cell Lines 6 Lysate Preparat lOt sus ves ccaessis cites cedescssdiact ca eiuasiess satevstaleiats uect ESEE 6 Considerations Before You Begin 6 Lysis Protocol for Cell Lines 7 Lysis Protocol for Tissue Samples 8 Flowchart for Lysate Preparation ccccccccsesscssessescssssseesseseeesseeaees 9 Bead based Immunoassay Protocol cccccccsesccessesscecseeseeeeseesees 10 Considerations Before You Begin 10 Step 1 Prepare Titration Buffer 10 Step 2 Prepare Standard Dilution Series 11 Step 3 Prepare Sample Dilutions 12 Step 4 Prepare Capture Beads 13 Step 5 Combine Capture Beads with Analytes 14 Step 6 Add Detection Antibodies 15 Step 7 Add Streptavidin Phycoerythrin PE 16 Flowchart for Breast Cancer Panel ImmunoasSa csecseeeees 17 Collecting Data and Data Analysis ccccccccscessseseecscsenetseeeceesenns 18 Data Acquisition 18 Generation of Standard Curves and Quantitation of Experimental Samples 18 Troubles noong kesane A 19 ADDONOIX EEE E E A A AE EAE EEEE 21 A Breast Cancer Panel Kits Ordering a
14. ach Antibody per well 5 pl total Volume 1X Assay Diluent 99 wl per well 95 ul per well 1 ul Antibody per well x 44 5 ul Antibodies per well x 44 wells Total Volume wells EE Detection Antibodies 100X 44 ul Detection 220 j E e Antibody p Total Volume 99 ul per well x 44 wells 95 ul per well x 44 wells 1X Assay Diluent 4356 ul 4180 ul 5 Remove liquid from filter plate by vacuum filtration 6 Add 100 ul 1X Wash Buffer to each well Remove liquid by vacuum filtration Repeat wash and filtration steps twice more for a total of three washes Tap filter plate on a paper towel to remove any buffer on the underside Do not allow the beads to dry out Vacuum only long enough to remove all liquid Add the next solution immediately after tapping filter plate on a paper towel 7 Immediately add 100 ul 1X Detection Antibody solution to each well Turn on the Luminex System The lasers require a 30 min warm up period 8 Incubate for 1 h at room temperature on a platform plate shaker 750 rpm Protect from light USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk All Other Countries www novagen com novatech novagen com User Protocol TB520 Rev A 0109 Page 16 of 25 Step 7 Add Streptavidin
15. al from an experimental sample exceeds the highest point of the standard curve the concentration of the unknown should not be extrapolated because the non linear shape of the standard curve cannot be accurately modeled past the last measured point In this case samples should be diluted and tested again USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 19 of 25 Troubleshooting Problem Probable Cause Solution Lysate is viscous Genomic DNA is not digested Make sure Benzonase Nuclease was added to Extraction Reagent Incubate lysate longer for eg 30 min For cell lines with recurring viscosity problems additional Benzonase Nuclease can be added available separately Leaking wells in filter plate Wicking due to adherent drops Tap filter plate several times on paper towel before adding samples or reagents Do not place filter plate on an absorbent surface during incubations If wells leaked during data acquisition it may be possible to reacquire these wells Blot underside of the wells and add 120 pl well 1X Assay Diluent Perforation of filter plate membranes Adjust the vacuum setting to lt 5 inches 127 m
16. ample Events per Timeout Doublet CAL2 Gain Size Bead Region Discriminator Setting Luminex 100 IS 50 pl 100 60 sec 7500 18500 default Bio Plex Manager Default 100 Default Default RP1 Low 50 ul 60 sec 4335 10000 If the time spent acquiring samples needs to be reducea collect as low as 50 events per bead region OR adjust the timeout as short as 30 sec Generation of Standard Curves and Quantitation of Experimental Samples e Standards are available for all of the Breast Cancer Panel Bead Kit see Appendix B allowing accurate quantification Representative standard curves and assay performance information can be found in the Certificates of Analysis for the individual bead kits e The 7 point standard curves are plotted using Median Fluorescent Intensity MFI as the signal readout Y axis against concentration of standard dilutions X axis Measurements of the blank are useful for assessing background and lower limits of detection However it is not necessary to subtract the MFI value of the blank from other measurements and the blank is generally not plotted as part of the curve e Five Parameter Logistic SPL curve fitting is recommended for modeling data Most ranges of standard curve concentrations are too wide for accurate linear regression analysis Four parameter 4PL equations will often give a good fit but are not ideal because they assume the standard curve is symmetrical e Ifthe sign
17. and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 14 of 25 4 Each well receives a total of 50 ul diluted 1X Capture Beads Determine the total volume of 50X Capture Beads needed per well refer to table above and the volume of 1X Assay Diluent needed to bring the total volume per well to 50 ul Multiply these volumes by the number of test wells to determine the total volumes of each component needed Refer to the table below for example calculations 5 Add the calculated volumes of Capture Beads 50X and 1X Assay Diluent to a microcentrifuge tube Vortex 3 sec Protect from light and store at 4 C until use Example Calculations 40 test wells including 10 extra Singleplex or User assembled Breast Cancer Panel I II or III Breast Cancer Panel multiplex multiplex premixed e g 5 Plex Test wells 40 44 including 10 extra 40 44 including 10 extra Volume Capture Beads 1 ul each bead per well 5 pl 1 ul per well 50X total Volume 1X Assay Diluent 49 ul per well 45 wl per well Total Volume 1 ul beads per well x 44 wells 5 pl beads per well x 44 wells Capture Beads 50X 44 ul beads 220 ul beads 44 ul ea Total Volume 49 ul per well x 44 wells 45 wl per well x 44 wells 1X Assay Diluent 21
18. ank prepared as per Step 2 Preparing Standard Dilution Series on p 11 6 To bead containing wells reserved for analyzing experimental samples add 100 ul diluted samples prepared as per Step 3 Prepare Sample Dilutions on p 12 If additional sample dilutions were prepared optional add 100 ul of these dilutions to bead containing wells Note If working with samples generated from cells grown in 96 well plates the four fold dilution with 1X Assay Diluent is done most easily at this step rather than as a separate step see Step3 Prepare sample dilutions To the appropriate bead containing wells add 75 ul 1X Assay Diluent and 25 ul clarified cell lysate using a multichannel pipet 7 Incubate overnight at 4 C on a platform plate shaker 750 rpm Use aluminum foil to protect filter plate from light Notes Shorter incubations are possible but will decrease overall signal strength USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 15 of 25 Make sure that the samples are not incubated below 4 C during this incubation step This can cause the lysates to acquire a gel like consistency and adversely affect results Note Note Note Step 6 Add Detect
19. ast Cancer Panels I II and IlI 5 Prepare 4 fold serial dilutions from Dilution 1 as follows Transfer 80 ul from tube 1 to the 240 ul titration buffer in tube 2 mix well Change tips Transfer 80 ul from tube 2 to the 240 ul titration buffer in tube 3 mix well Proceed in similar manner with the serial dilutions through tube 7 6 The 8th tube contains 240 ul titration buffer only This will serve as the blank control Note Refer to Appendix B on p 23 for concentrations of the serially diluted standards USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 12 of 25 Table 3 Serial dilution of singleplex or pre mixed Breast Cancer Panel I II and III standards mix Tube Volume Standard Volume Final Concentration Dilution Titration Buffer 1 30 ul individual Standard or Breast Cancer 270 ul Panel I II or III Standards Mix 2 80 ul from tube 1 240 ul 3 80 ul from tube 2 240 ul 4 80 ul from tube 3 240 ul pee Apperidi B 5 80 ul from tube 4 240 ul 6 80 ul from tube 5 240 ul 7 80 ul from tube 6 240 ul 8 BLANK None 240 ul 0 Table 4 Example of serial dilution of five individual Breast Cancer Panel Standards
20. ation Use BCA Protein Assay Proceed to bead based immunoassay or store lysate aliquots at 70 C USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de All Other Countries www novagen com novatech novagen com United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 10 of 25 Bead based Immunoassay Protocol Note Considerations Before You Begin e Have on hand the 1X Assay Diluent and 1X Wash Buffer that was prepared during the Lysate Preparation protocol e Important guidelines to follow when using filter plates and the vacuum manifold e Excessive vacuum will cause the filter plate membrane to perforate Adjust the manifold using a non filter ELISA or tissue culture plate ensuring that the vacuum cannot exceed 5 in 127 mm Hg e After adjusting the vacuum place filter plate on the manifold Use fingertips to apply pressure evenly across the plate The liquid should drain in 2 5 sec e To avoid drying out the beads vacuum only long enough to drain all wells Do not allow drained beads to sit for more than 1 min before rehydrating with buffer e It is critical to remove excess buffer from the underside of the filter plate by tapping it on a paper towel several times before adding samples or reagents This prevents samples from wicking out of t
21. atios have been shown to regulate the invasive potential of breast tumors Components WideScreen Breast Cancer Panel Bead Kits are used for quantitative multiplex analysis of cell culture lysates and normal or tumor tissue lysates The WideScreen Breast Cancer Panel I II and I Complete Kits contain sufficient reagents to assay one 96 well plate For maximum flexibility and user defined multiplex assay configuration the components of the WideScreen Breast Cancer Panel I I and I Complete Kits are available separately WideScreen Breast Cancer Panel I II and III Complete Kits The WideScreen Breast Cancer Panel I II or I Complete Kits include the entire set of reagents to assay one 96 well plate including the Breast Cancer Panel I II or HI multiplex Breast Cancer Panel I II or IN Standards Mix Cell Extraction Kit and WideScreen Reagent Kit Breast Cancer Panel I II and Ill Breast Cancer Panel I II and III are premixed panels that contain premixed antibody coated Capture Beads biotinylated Detection Antibodies and premixed recombinant standards used for target quantification via multiplex sandwich immunoassay Each Breast Cancer Panel contains sufficient reagents to assay one 96 well plate Breast Cancer Panel I II and III require the purchase of the Cell Extraction kit and the WideScreen Reagent Kit Target analytes for the premixed panels are listed in Table 1 Appendix A on p 21 and at www novagen com WideScreen Eac
22. d Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 11 of 25 1 Calculate the total amount of Titration Buffer needed A minimum of 2000 wl titration buffer is neededto prepare a duplicate standard curve see Step 2 Prepare Standard Dilution Series below A minimum of 300 pl titration buffer is needed for each lysate sample that is diluted more than 4 fold final see optional steps in Step 3 Prepare Sample Dilutions on p 12 Sample Calculation 2 Standard dilution series 2000 pl 30 diluted lysate samples 9000 ul 30 X 300 ul Make at least 11000 ul titration buffer 2 Prepare the required volume titration buffer by mixing Extraction Reagent from the Cell Extraction Kit and 1X Assay Diluent prepared from the WideScreen Reagent Kit Use a ratio of 25 Extraction Reagent to 75 1X Assay Diluent In the example above take 2750 ul Extraction Reagent 8250 ul 1X Assay Diluent 11000 ul Titration Buffer allowing for additional buffer to account for pipetting error Step 2 Prepare Standard Dilution Series Prepare fresh diluted standards for each assay and use within 1 h 1 To prepare duplicate 7 point standard curves label eight microcentrifuge tubes and add 240 ul Titration Buffer to tubes 2 8 See Step 1 Prepare Ti
23. ests Components and Storage Cell Extraction Kit 71926 3 25 ml Extraction Reagent Store at 20 C 500 pl Phosphatase Inhibitor Cocktail Set V Store at 20 C 50x 25ul Protease Inhibitor Cocktail Set II Store at 20 C 1000x 10 pl Benzonase Nuclease HC Purity Store at 20 C gt 99 250 U pl WideScreen Reagent Kit 71783 3 100 ul Streptavidin PE Concentrate Store at 4 C 20 ml 10X Wash Buffer Store at 4 C 25 ml 5X Assay Diluent Store at 4 C lea Polyethylene Plate Sealer Store at room temperature lea 96 well Filter Plate Store at room temperature Components and storage conditions for WideScreen Breast Cancer Panel Complete Kits and Breast Cancer Panel I II and II Multiplex and Individual Bead Kits are described in Appendix A on p 21 Additional Materials Required But Not Supplied e Experimental samples such as cultured cell lines or tissue samples e Dounce Homogenizer or Micro dismembrator for preparing tissue lysates e Luminex System or comparable such as Bio Plex Suspension Array System e xMAP data analysis software e g Luminex IS ACS StarStation Bio Plex Manager or comparable e Vacuum manifold for filter plates Pall 5017 or Millipore Cat No MAVMO0960R e 96 well plate platform shaker such as IKA MTS4 e BCA protein assay kit Novagen Cat No 71285 e Polypropylene microcentrifuge tubes e 15 mland 50 ml p
24. ge effects don t plate cells in outermost wells of plates Plate cells uniformly Add lysis reagents accurately Do not dislodge adherent cells during pre lysis wash steps If necessary decant instead of aspirating liquid and tap plate on paper towels If cells become less adherent during overnight serum starvation shorten the serum starvation step to 4 h A gradual drop in signal strength as many samples are read on the xMAP system Group samples such that those being compared directly including replicates are being read without a long delay in between Use 0 2 paraformaldehyde in TBS to covalently fix PE to bead surfaces Lysates assayed at different times show assay to assay variability Generate standard curves carefully using at least duplicate dilutions series to increase inter assay precision Fully resuspend standards and lysate samples by thawing to room temperature and vortexing carefully Sample measurements not falling on the standard curve Dilution of lysate is too low or too high If values are higher than the standard curve dilute samples further in titration buffer Signal strength may be boosted by increasing lysate protein concentration by lysing cells at a higher confluence or by using less Extraction Reagent Standard curve and background values increased due to multiplexing The standard curves of some assays shift slightly upon multiplexing Therefore for accurate quanti
25. h premixed standards mix contains reagents sufficient to generate eight multiplex standard curves or four standard curves in duplicate The concentration of premixed standards in the standard curves can be found in Appendix B on p 23 and at www novagen com WideScreen Performance specifications for the Multiplex Kits are detailed in the Certificates of Analysis available online Individual Breast Cancer Panel I II and Ill Bead Kits Individual Bead Kits for each analyte for setting up singleplex or user defined multiplex experiments may be purchased separately These kits include antibody coated Capture Beads biotinylated Detection Antibody and the appropriate individual recombinant standard used for target detection via sandwich immunoassay Each individual Bead Kit contains sufficient reagents to assay one 96 well plate Individual Bead Kits require the purchase of the Cell Extraction kit and the WideScreen Reagent Kit Available Individual Bead Kits are listed in Appendix A on p 21 and at www novagen com WideScreen The concentration of each standard can be found in Appendix B on p 23 and at www novagen com WideScreen Each individual standard contains reagents sufficient to generate eight singleplex standard curves or four standard curves in duplicate Performance specifications for the Individual Bead Kits are detailed in the individual Certificates of Analysis available online Individual Breast Cancer Panel Bead Kits and buffer
26. he wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations e To avoid perforating the filter plate membrane be sure that the probe height on the Luminex System is adjusted correctly Do not touch the membrane with pipet tips For accurate pipetting touch tips to the sides of the filter plate wells Change tips as necessary to prevent cross contamination e Capture Beads contain fluorescent dyes and are therefore light sensitive To avoid photobleaching keep beads in microcentrifuge tubes covered Cover filter plates containing beads with aluminum foil during incubation steps Streptavidin PE solution is also light sensitive protect from light e To prevent fluorescent dye loss do not use organic solvents with capture beads Beads are incompatible with DMSO concentrations gt 1 e Many of the liquid handling steps such as washing are done most easily with an 8 channel or 12 channel pipet manual or automatic However for best results use accurate single channel pipets for manipulation of standards and experimental samples e Ifusing multichannel pipets ensure that tips fit correctly Verify volume accuracy and consistency e To conduct the protocol efficiently prepare reagents for the next step during sample incubation period e When calculating the amount of reagents needed during the various steps prepare 10 excess to allow for pipetting error e Run standard d
27. ilution series and experimental samples using the same multiplex configuration For instance if a 6 Plex of Bead Kits is used to measure experimental samples the same 6 Plex should be used to create the standard dilution series Multiplexing causes slight shifts in some standard curves which will make quantification inaccurate unless experimental samples are measured using the same multiplex e For best overall assay performance lysates are diluted at least 4 fold when incubating with the Capture Beads If desired lysates can be tested at a 2 fold final dilution although this concentration of Lysis Buffer decreases the sensitivity of some Bead Kits If a 2 fold final dilution is used change the titration buffer composition to 50 Lysis Buffer 50 1X Assay Diluent to ensure accurate quantification Final dilutions less than 2 fold are not recommended Step 1 Prepare Titration Buffer Quantitative immunoassays are sensitive to buffer composition Therefore include the same proportion of Extraction Reagent in all dilutions of standards and samples The best overall assay performance occurs when lysates are diluted at least 4 fold when incubated with the Capture Beads Titration buffer as described here 25 Extraction Reagent 75 1X Assay Diluent maintains a 4 fold final dilution of Extraction Reagent in all assay wells Prepare fresh titration buffer for each assay USA and Canada Tel 800 526 7319 novatech novagen com Germany Unite
28. ion Antibodies Prepare 1X Detection Antibody solution within 1 h of use Calculate the number of test wells needed 2 Note the volume of 100X Detection Antibody needed per well based on the assay format see table below Assay Format Volume Detection Antibodies 100X needed Singleplex one target 1 pl per well User assembled Breast Cancer Panel multiplex 1 ul from each individual Bead Kit per well Breast Cancer Panel x Plex premixed 1 ul per well 3 Each well receives a total of 100 ul diluted 1X Detection Antibody solution Determine the total volume of 100X Detection Antibodies needed per well refer to the table above and the volume of 1X Assay Diluent needed to bring the total volume per well to 100 ul Multiply these volumes by the number of test wells to determine the total volumes of each component needed Prepare 10 extra for pipetting error Refer to the table below for example calculations 4 Add the calculated volumes of Detection Antibodies 100X and 1X Assay Diluent to a microcentrifuge tube Vortex 3 sec and store at 4 C until use Example Calculations 40 test wells including 10 extra Singleplex or Breast Cancer Panel I II or ITI Multiplex premixed User assembled Breast Cancer Panel multiplex e g 5 Plex Test wells 40 44 including 10 extra 40 44 including 10 extra Volume Detection Antibodies 100X 1 pl per well 1 pl e
29. ion beads Verify correct system pressure Confirm that the system is free of air or particulate buildup Follow maintenance steps described in the instrument user manual All Other Countries www novagen com novatech novagen com USA and Canada Tel 800 526 7319 novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 20 of 25 Problem Probable Cause Solution An immunoassay reagent is used up Solutions were not prepared or used as described in the protocol Briefly spin tubes to collect reagents that might be trapped in the tube cap Confirm correct buffer dilutions and use If additional Wash Buffer is needed TBST 10 mM Tris pH 7 5 150 mM NaCl 0 05 Tween 20 may be substituted If additional Assay Diluent is needed 10 mM Tris pH 7 5 225 mM NaCl 0 05 Tween 20 1 BSA may be substituted If additional 96 well filter plates are required we recommend Millipore Cat No MSBVN1210 If there is insufficient volume of 100X Streptavidin PE for your final experiment making a slightly more dilute working stock e g 0 75X instead of 1X will not adversely affect results High coefficients of variance CVs between replicates Cells grown in 96 well plates show well to well variability To avoid ed
30. is A primary player is the receptor protein VEGFR 2 that is expressed on breast tumor tissue with its ligand VEGF A Other pro angiogenic molecules involved in breast cancer include angiogenin USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 4 of 25 Note Note Note inducing neovascularization and angiopoietin 2 affecting vascular remodeling via integrin and E cadherin signaling pathways While vascular remodeling is important for solid tumor growth it also contributes to the spreading of the primary tumor to secondary sites metastasis a leading cause of poor breast cancer prognosis Several extracellular matrix remodeling activities are required for tumor invasion and metastasis An example of a proteolytic protein involved in breast cancer invasion and metastasis is urokinase type plasminogen activator uPA and its inhibitor plasminogen activator inhibitor 1 PAI 1 that affect fibrinolysis and cell migration In addition proteolytic enzymes such as matrix metalloproteinases MMPs and their endogenous inhibitors tissue inhibitors of metalloproteinase TIMPs mediate extracellular matrix remodeling Specifically MMP 9 TIMP 1 and MMP 2 TIMP 2 r
31. ix Final concentrations in the 4 fold serial dilution of the standards Angiopoietin 2 PR HER 2 PAI 1 IGF 1R Angiogenin Mol Wt kDa 66 102 96 50 48 14 Bead Region 14 22 72 20 25 13 pg ml pg ml pg ml pg ml pg ml pg ml Dilution 1 10000 3000 20000 20000 100000 200 Dilution 2 2500 750 5000 5000 25000 50 Dilution 3 625 188 1250 1250 6250 13 Dilution 4 156 47 313 313 1563 3 Dilution 5 39 12 78 78 391 0 8 Dilution 6 10 3 20 20 98 0 2 Dilution 7 2 5 0 7 5 5 24 0 1 Blank 0 0 0 0 0 0 Breast Cancer Panel II Standards Mix Final concentrations in the 4 fold serial dilution of the standards EGFR Fas VEGFR 2 ERa TIMP 2 Mol Wt kDa 68 45 160 66 22 Bead Region 21 24 76 17 18 pg ml pg ml pg ml pg ml pg ml Dilution 1 20000 2000 100000 50000 30000 Dilution 2 5000 500 25000 12500 7500 Dilution 3 1250 125 6250 3125 1875 Dilution 4 313 31 1563 781 469 Dilution 5 78 8 391 195 117 Dilution 6 20 2 98 49 29 Dilution 7 5 0 5 24 12 7 Blank 0 0 0 0 0 USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 24 of 25 Breast Cancer Panel III Standards Mix Final concentrations in the 4 fold serial dilution of the standards
32. l Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 22 of 25 WideScreen Breast Cancer Panel I Complete Kit 72088 3 1 Breast Cancer Panel 6 Plex which includes e Breast Cancer Panel I Capture Beads Premix 6 Plex e Breast Cancer Panel Detection Antibody Premix 6 Plex e Breast Cancer Panel I Standards Mix 6 Plex Store at 4 C Cell Extraction Kit see p 5 for components Store at 20 C WideScreen Reagent Kit see p 5 for components see p 5 for storage conditions WideScreen Breast Cancer Panel II Complete Kit 72089 3 1 Breast Cancer Panel 5 Plex which includes e Breast Cancer Panel II Capture Beads Premix 5 Plex e Breast Cancer Panel Detection Antibody Premix 5 Plex e Breast Cancer Panel II Standards Mix 5 Plex Store at 4 C Cell Extraction Kit see p for components Store at 20 C WideScreen Reagent Kit see p 5 for components see p 5 for storage conditions WideScreen Breast Cancer Panel III Complete Kit 72090 3 1 Breast Cancer Panel 4 Plex which includes e Breast Cancer Panel III Capture Beads Premix 4 Plex e Breast Cancer Panel Detection Antibody Premix 4 Plex e Breast Cancer Panel III Standards Mix 4 Plex Store at 4 C Cell Extraction Kit see p 5 for components Store at 20 C WideScreen Reagent Kit see p for components
33. l sample of each extract for protein quantification by BCA Protein Assay Cat No 71285 Determine the total protein concentration of each extract 9 Either proceed immediately to the Bead based Immunoassay Protocol on p 10 or store aliquots at 70 C Avoid multiple freeze thaw cycles Tissue homogenization and lysate preparation using Micro dismembrator For this protocol a Micro dismembrator bead or ball mill is recommended Flash frozen tissue samples should be used This protocol has been successfully applied to breast tissue using a 1 10 w v ratio of tissue to Supplemented Extraction Reagent Other tissue types may require ratio optimization 1 Prepare the required volume of supplemented Extraction Reagent Per ml Extraction Reagent add 1 pl Protease Inhibitor Cocktail III 1000X 0 1 ul Benzonase Nuclease 20 ul Phosphatase Inhibitor Cocktail Set V 50X Notes Prepare fresh supplemented Extraction Reagent each time cell lysates are made Because the Breast Cancer Panel assays are not phospho specific the use of Phosphatase Inhibitor Cocktail Set V is optional 2 Determine tissue weight Transfer aliquots of 30 60 mg tissue to a tube and keep samples frozen normal tissue 60 mg tumor tissue 30 mg Freeze accessories for Micro dismembrator with liquid nitrogen 4 Add 10 parts Supplemented Extraction Reagent for each part tissue e g 500 ul supplemented Extraction Reagent per 50 mg tissue into the dismembrator tube
34. ll filter plate e g Millipore Cat No MSDVN6510 and centrifuge at 1500 x g for 1 min at 4 C Place a 96 well plate under the filter plate during centrifugation to collect lysates 10 Remove a 50 ul sample of each extract for protein quantification by BCA Protein Assay Cat No 71285 Determine the total protein concentration of each extract Note Typical total protein concentrations from cells grown in flasks range from 0 4 mg ml to 2 mg ml depending on the cell line and confluence Typical total protein concentrations from cells grown in 96 well plates range from 0 1 0 5 mg ml 11 Either proceed immediately to the Bead based Immunoassay Protocol on p 10 or store aliquots at 70 C Avoid multiple freeze thaw cycles USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 8 of 25 Lysis Protocol for Tissue Samples Tissue homogenization and lysate preparation using Dounce Homogenizer For this protocol a Dounce Homogenizer is recommended Fresh or flash frozen tissue samples can be used This protocol has been successfully applied to breast tissue using a 1 15 w v ratio of tissue to supplemented Extraction Reagent Other tissue types may require ratio o
35. m Hg Do not touch membranes with pipet tips Filter plate wells not draining under vacuum Vacuum is too low Adjust vacuum setting to 3 5 inches 76 127 mm Hg Clean rubber seals Apply fingertip pressure to filter plate to ensure formation of a good seal Use a plate sealer to cover wells not in use Cell debris clogs membranes Clarify lysates by centrifugation Avoid disturbing pellets when removing supernatant Use the non tip end of a fresh 200 ul pipette tip to flick the center support on the underside of the well then reapply vacuum If lysate protein concentration is high dilute further before assaying Low bead counts during data acquisition No beads or wrong beads in the wells See solutions above for leaking wells Verify that the appropriate beads were added at the correct concentration and that the correct bead regions and wells were selected during acquisition setup Luminex fluidics system is clogged Clear system of clogs or air using maintenance steps described in the instrument user manual sanitize alcohol flush probe sonication etc Make sure that the probe height is set correctly Make sure that beads are in suspension by incubating plate for 3 5 min on the platform plate shaker 750 rpm immediately before analysis Microbial growth in buffers can cause beads to stick to the filter plate membrane Do not use contaminated reagents Timeout limit is set too l
36. ncer Panel I II and III analytes are directly or indirectly implicated in breast cancer biology including tumor growth apoptosis angiogenesis and metastasis These include receptor proteins and a myriad of signaling and effector proteins that have the ability to crosstalk with each other The levels of many of these proteins in breast tumor tissue differ when compared to adjacent normal tissue Identifying these proteins can assist in the understanding of the tumor microenvironment and the fundamental biology of tumor formation Expression profiling of extracellular receptor tyrosine kinases such EGFR and HER 2 and intracellular steroid receptors such as estrogen receptor a and progesterone receptor in breast tumor tissue has resulted in tumor classification that aids in breast cancer treatment and prevention Another receptor tyrosine kinase IGF 1R is known to promote growth transformation and antiapoptotic activity of breast tumor tissue IGF 1R activity is further controlled by binding proteins such as IGFBP 3 that bind to IGFs with a greater affinity compared to the IGF 1R receptor Additional signaling molecules are involved in controlling the apoptosis of the breast cancer cells For instance the expression levels of Fas receptor and its ligand FasL on breast tumor tissue regulate Fas mediated apoptosis in breast cancer cells Breast tumor growth is promoted by proteins that are involved in new blood vessel formation i e angiogenes
37. nd Storage Information 21 B Dilution Series for Generating Standard Curves 23 C WideScreen Breast Cancer Panels Assay Compatibility Chart 25 Europe France Germany Ireland United Kingdom All other Freephone Freecall Toll Free Freephone European Countries 0800 126 461 0800 1003496 1800 409 445 0800 622 935 44 115 943 0840 techservice merckbio eu www novagen com FOR RESEARCH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE All Other Countries Contact Your Local Distributor www novagen com novatech novagen com User Protocol TB520 Rev A 0109 Page 2 of 25 2009 EMD Chemicals Inc an Affiliate of Merck KGaA Darmstadt Germany All rights reserved The Novagen name and Novagen logo are registered trademarks of EMD Chemicals Inc in the United States and in certain other jurisdictions WideScreen is a trademark of EMD Chemicals Inc Benzonase is a registered trademark of Merck KGaA Darmstadt Germany Bio Plex is a registered trademark of Bio Rad Laboratories Inc Luminex and xMAP are registered trademarks and Luminex 100 IS and Luminex 200 are trademarks of Luminex Corporation By opening the packaging containing this product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute
38. ng to final volume of 300 ul with buffer to final volume of 400 pl titration buffer 150 ul titration buffer in tubes 2 3 240 ul titration buffer in tubes 2 7 2 fold serial dilutions 150 pl from 4 fold serial dilutions 80 ul from tube tube 1 to tube 2 etc 1 to tube 2 etc Pre wet Filter Plate 50 ul 1X Assay Diluent per well vacuum filter after 5 min Capture Bead Analyte Incubation 50 ul diluted 1X Capture Beads to all wells Remove liquid by vacuum filtration Add 100 ul sample dilutions to beads in appropriate wells Add 100 ul standard dilutions to beads in appropriate wells Add 100 ul titration buffer to beads in Blank well s Shake overnight 750 rpm 4 C protected from light Detection Antibody Incubation Per well 1 ul Detection Antibody 100X from each bead kit 1X Assay Diluent to final volume of 100 ul Wash amp vacuum filter plate 3 times 100 pl 1X Wash Buffer per well Add 100 ul diluted 1X Detection Antibody mix to each well Shake 1 h 750 rpm room temperature protected from light Streptavidin PE Incubation Per well 1 pl Streptavidin PE Concentrate 99 ul 1X Assay Diluent Wash amp vacuum filter plate 3 times 100 ul 1X Wash Buffer per well Add 100 ul diluted Streptavidin PE to each well Shake 45 min 750 rpm room temperature in darkness Optional Add 30 ul fixation solution to each well 0 2 paraformaldehyde in TBS not provided in the kit Shake 5 min 750 rpm room temperature protected fr
39. olypropylene centrifuge tubes e Microcentrifuge e Vortexer e Ultrasonic bath such as Cole Parmer EW 08849 optional e Multichannel pipet optional e Fixation solution 0 2 paraformaldehyde in PBS optional e Syringe tip filter 0 45 um and syringe or 96 well filter plate e g Millipore Cat No MSDVN6510 and 96 well collector plate e Tris buffered saline TBS 10 mM Tris pH 7 5 150 mM NaCl USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 6 of 25 Growth of Cell Lines Considerations Before You Begin e Growth rate and requirements for optimal growth vary considerably between cell lines even the same cell line will grow differently in different laboratories The following conditions are intended as general guidelines only e Cells maintained in culture for long periods of time tend to exhibit slower growth rates and become refractory to stimulation conditions In general cell lines passaged lt 15 times are recommended Protocol for Growth of Cell Lines 1 Culture cells in T 75 flasks until steady growth is established Most cell lines will tolerate a split of 1 10 1 20 without slowing their growth rate 2 Culture adherent cells un
40. om light Analysis Wash amp vacuum filter plate 3 times 100 ul 1X Wash Buffer per well Add 120 ul 1X Assay Diluent to each well Shake 5 min 750 rpm room temperature in darkness Run on Luminex system Low Gain RP1 setting BioPlex DD Gate 7500 18500 Luminex Sample size 50 ul Collect 100 events per bead region Timeout 60 sec USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 18 of 25 Collecting Data and Data Analysis Data Acquisition For detailed instructions on the operation of Luminex Systems refer to the user guide for your specific software and instrument General recommendations are given below 1 Using your Luminex System software prepare a protocol for the assay you will run Enter in information for each bead kit target standards samples and controls that will be run The ranges of final concentrations are shown in Appendix B 2 Select the bead regions used in the assay The bead regions used for the Breast Cancer Panel Bead Kits are shown in Appendix B Format the assay plate indicating which wells contain which type of analyte 4 Acquire data using the system settings shown below Software S
41. ore size 0 45 um For lysates with volume lt 0 2 ml use a 96 well filter plate e g Millipore Cat No MSDVN6510 centrifuge 1500 x g 1 min 4 C Prepare Stock Solutions 1X Assay Diluent Dilute 5X five fold with water 1X Wash Buffer Dilute 10X ten fold with water Prepare Supplemented Extraction Reagent per 1 ml Extraction Reagent e Phosphatase Inhibitor Cocktail Protease Inhibitor Cocktail e Benzonase Nuclease 20 ul Tissue Samples Homogenizer Tissue samples Micro dismembrator bead mill Cell Lysis Cell Lysis Determine tissue weight Transfer tissue samples 50 120 mg to tubes Add 15 parts v w Supplemented Extraction Reagent Determine tissue weight Transfer tissue samples 30 60 mg to tubes Cool micro dismembrator accessories with liquid nitrogen Add 10 parts v w Supplemented Extraction Reagent into Dismembrator tube and let it freeze Add tissue and steel ball Pulverize for 1 min at 2000 rom Incubate for 60 min on ice Disrupt samples by using a homogenizer Incubate for 30 min on ice Clarify tissue lysates Centrifugation of tissue lysates at 16000 x g 10 min 4 C Transfer supernatant to new tube Repeat centrifugation step Transfer supernatant to new tube Clarify tissue lysates Centrifugation of tissue lysates at 16000 x g 10 min 4 C Transfer supernatant to new tube Repeat centrifugation step Transfer supernatant to new tube Determine Total Cellular Protein Concentr
42. ot meant to represent assay cross recognition or target specificity For information on assay specificity see the Certificates of Analysis for individual Breast Cancer Panel Bead Kits IGF 1R Compatible Progesterone P receptor Compatible with increased background HER 2 Not Compatible PAI 1 Angiopoietin 2 EGFR TIMP 2 VEGFR 2 Fas ERa TIMP 1 uPA E Cadherin IGFBP 3 l z E c p T D 2 N D 2 BSa 2 a oo Dene ae ee s g 8 2 y Su z G6 Zo ec 2 EE YO lt x a SO as alo ai lt W e gt IL W H 3 W USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk
43. ow Use the recommended settings for acquisition setup first 50 ul sample 100 events per bead 60 sec time out etc However timeout limit can be set higher e g 75s Data acquisition is slow No beads in the wells or fluidics system is clogged See Low bead counts during data acquisition solutions above Some bead regions being acquired are not in the wells Make sure that the intended beads were added and that the correct bead regions and wells were selected during acquisition setup Attempting to acquire inappropriate bead regions will cause each sample to time out Beads are not falling into the gates properly Beads were not resuspended in 1X Assay Diluent before analysis The setting of the Doublet Discriminator DD gate is buffer specific This gate can be adjusted but 1X Assay Diluent is the buffer recommended for running samples Other buffers may also cause bead aggregation Beads were exposed to organic solvents Do not use organic solvents in the immunoassay as they will damage beads irreversibly Beads are falling outside the bead region gates due to photobleaching Do not use expired beads Do not expose the beads to ambient light for gt 10 min Avoid intense light Fluidics system is not running properly Confirm that the waste container is not full the sheath fluid is not empty and the SD fluidics module is turned on Check system calibration using approved calibrat
44. ptimization For homogenization use a tube large enough to accommodate some foaming 1 Calculate the total amount of Extraction Reagent needed Prepare 10 excess to account for pipetting error 2 Prepare the required volume of supplemented Extraction Reagent Per ml Extraction Reagent add 1 pl Protease Inhibitor Cocktail III 1000X 0 1 ul Benzonase Nuclease 20 ul Phosphatase Inhibitor Cocktail Set V 50X Notes Prepare fresh supplemented Extraction Reagent each time tissue lysates are made Because the Breast Cancer Panel assays are not phospho specific the use of Phosphatase Inhibitor Cocktail Set V is optional 3 Determine tissue weight Transfer aliquots of 50 120 mg tissue to 5 ml round bottom polypropylene tubes Keep samples on ice normal tissue 120 mg tumor tissue 50 mg 4 Add 15 parts v w room temperature supplemented Extraction Reagent for each part tissue e g 750 ul supplemented Extraction Reagent per 50 mg tissue Immediately return sample to ice 5 Homogenize with a hand held homogenizer or other suitable device Homogenization time will vary with tissue type but 1 minute is generally required for efficient protein extraction Return sample to ice Incubate for 30 min on ice with occasional vortexing Clarify lysate by centrifuging twice at 16 000 x g for 10 min at 4 C After each centrifugation transfer supernatants to new microcentrifuge tubes after each centrifugation step 8 Remove a 50 u
45. s beads covalently conjugated to a capture antibody Analytes captured on the beads are identified with detection antibodies and a fluorescent label A major hallmark of bead based assays over traditional protein quantitation methods such as ELISA is the capacity for multiplexing as bead based assays allow simultaneous quantitation of multiple analytes in a small sample volume Other advantages of xMAP assays include flexibility robustness simple sample handling and requirement of minimal sample volumes making them an ideal platform for immunodetection of biomarkers and signaling proteins WideScreen Breast Cancer Panels I II and III are premixed multiplex bead kits for simultaneous quantitation of proteins reported to be involved in breast cancer see Table 1 for a complete list of analytes The bead kits are available as a choice of three multiplex panels and individually facilitating the creation of singleplex and custom multiplex experiments WideScreen Breast Cancer Panel I II and III Complete Kits include all the reagents and buffers needed to analyze the proteins listed below each respective panel see Table 1 in cell culture lysates and normal or tumor tissue lysates using the Luminex xMAP System Table 1 Target recognition for Breast Cancer Panels I II and III Species specificity Human other species not tested For more information on Widescreen Bead based Assays visit www novagen com WideScreen Breast Ca
46. s are not necessarily compatible with other bead kits and reagents sold by Novagen or other vendors Please refer to Appendix C on p 25 for compatibility of assays found in WideScreen Breast Cancer Panels I II and IlI USA and Canada Tel 800 526 7319 novatech novagen com Germany United Kingdom and Ireland All Other Countries Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com techservice merckbiosciences de Ireland Toll Free 1800 409445 novatech novagen com customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 5 of 25 Cell Extraction Kit The Cell Extraction Kit contains a cell extraction reagent that releases soluble and membrane proteins efficiently Benzonase Nuclease included reduces viscosity due to chromosomal DNA Phosphatase and protease inhibitor cocktails included maintain the phosphorylation state and integrity of target proteins during cell extraction The kit contains reagents sufficient to make 20 ml of cell lysate or to process 160 wells of cells grown in 96 well plates Additional extraction reagent is included for preparation of titration buffer WideScreen Reagent Kit The WideScreen Reagent Kit contains reagents needed for the bead based immunoassays including all buffers a 96 well Filter Plate a Plate Sealer and a Streptavidin Phycoerythrin solution used in the final detection step The kit contains sufficient reagents to perform 96 singleplex or multiplex bead based t
47. tation the same multiplex of assays must be prepared when comparing standard curves and experimental samples Target concentration is below detection Not all analytes are expressed detectably in all cell lines and tissue lysates Screen additional cell lines Target expression may be suboptimal in some cell lines or tissue samples Confirm that antibodies used in the assay recognize target in the species being tested USA and Canada Tel 800 526 7319 novatech novagen com All Other Countries www novagen com novatech novagen com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB520 Rev A 0109 Page 21 of 25 Appendix A Breast Cancer Panel Kits Ordering and Storage Information Each Bead Kit contains the following components e 100 ul Capture Bead 50X use 1 ul per test e 100 ul Detection Antibody 100X use 1 ul per test e 120 ul Breast Cancer Panel Standard s 10x lyophilized Note Individual Breast Cancer Panel Bead Kits can not be multiplexed together in all combinations because of incompatibilities between some assays Please see Appendix C on p 25 for further information Breast Cancer Panel I 6 Plex 100 tests Angiopoietin 2 progesterone receptor
48. til they approach a confluent monolayer or suspension cells until they approach 10 cells per ml Slower growing cell lines such as A431 may initially take up to a week to approach confluency 3 Plate cells using the following table as a general guide Harvest cells for lysate preparation after 2 or 3 days depending on whether the cells are serum starved overnight before harvesting Table 2 Approximate Cell Numbers for Seeding Cell Lines Cell Line apes VA AUN her 96 well Plate per well or 10 cm Dish well T47D 2 4x 10 3 4x 10 3 0 x 10 MCE 7 1 2x 10 1 7x 10 1 5x 10 BT20 1 2x 10 1 7x 10 1 5x 104 A431 2 0 x 10 2 8 x 10 4 0 x 10 HeLa 1 2x 10 1 7x 10 1 5x 10 HepG2 4 8 x 10 6 8 x 10 8 0 x 104 HT29 2 4x 10 3 4x 10 3 0 x 104 HUVEC 1 5 x 10 2 0 x 10 not recommended NHDF 15x 10 15x 10 not recommended SK Br 3 2 0 x 10 2 8x 10 3 0 x 10 Jurkat 1 0 x 10 1 4x 10 1 5 x 104 HUVEC and NHDF cells plated with these cell numbers are serum starved after 6 days and lysed after 7 days Note If cells are grown in 96 well plates plate extra wells for determining total protein concentration of the lysates 4 Prepare lysates when cell density is high but cells are still growing logarithmically For adherent cells this is typically a monolayer that is 80 confluent For suspension cells this is typically a density of 0 5 1 0 x 10 per ml
49. tration Buffer on p 10 2 Resuspend the appropriate lyophilized Breast Cancer Panel Standards in 120 ul titration buffer for each analyte being tested These represent 10X Standard solutions Vortex briefly to ensure all standards are fully resuspended 3 Ifconducting a singleplex or using the Breast Cancer Panel I II or IN Standards Mix add 30 ul of the standard or the premixed multiplex standards respectively to 270 ul Titration Buffer for a final volume of 300 pl This tube is Dilution 1 of the standard dilution series Please refer to Table 3 for the serial dilution series 4 Ifconducting a user assembled multiplex assay add 30 ul of each of the individual Breast Cancer Panel Standards 10X being assayed to tube 1 Bring the total volume of tube 1 to 300 ul with Titration Buffer and mix well This tube is Dilution 1 of the standard dilution series Please refer to Table 4 on the next page for an example of the serial dilution series for a user assembled multiplex Notes The volume of titration buffer added to reconstitute the 300 pl in a user assembled multiplex will depend upon the number of analytes included in your multiplex Please refer to Table 4 on the next page for an example The WideScreen Breast Cancer Panel Bead Kits and buffers are not necessarily compatible with other bead kits and reagents sold by Novagen or other vendors Please refer to Appendix C on p 25 for compatibility of assays found in WideScreen Bre
50. user assembled 5 Plex Tube Vol Standard Volume Final Concentration Dilution Titration Buffer 1 5 x 30 ul of each individual Breast Cancer 150 ul Panel Standard 10X 150 ul total volume 2 80 ul from tube 1 240 ul 3 80 ul fi tube 2 240 ul ore ne 3 See Appendix B 4 80 ul from tube 3 240 ul 5 80 ul from tube 4 240 ul 6 80 ul from tube 5 240 ul 7 80 ul from tube 6 240 ul 8 BLANK None 240 ul 0 Note Individual Breast Cancer Panel Bead Kits can not be multiplexed together in all combinations because of incompatibilities between some assays Please see Appendix C on p 25 for further information Step 3 Prepare Sample Dilutions Notes Thaw and dilute samples within 1 h of use Avoid multiple freeze thaw cycles For lysate samples prepared from cells grown in 96 well plates typical total protein concentrations range from 0 1 0 5 mg ml Therefore 96 well lysate samples can be diluted directly at the time that the samples are added to the capture beads Further sample dilutions might not be required see Step 5 Combine Capture Beads with Analytes on p 14 1 Dilute lysate samples four fold in 1X Assay Diluent e g 100 ul lysate with 300 ul 1X Assay Diluent Mix well USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 526 7319 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com novatech novagen com techservice merckbiosciences de Ireland Toll Free 1800

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