Instruction manual - Bio-Rad
Contents
1. If using diluents other than 10 serum based standard diluent users must establish their own control ranges At least 0 5 final BSA is recommended to stabilize analytes and reduce adsorption to labware 12 Reconstitute Standards and Controls This procedure prepares enough standard to run each dilution in duplicate Note The appearance of the lyophilized standards or controls may vary from a white pellet to clear crystals Regardless of appearance the vials have passed QC specifications and perform accordingly ill Gently tap the vial containing the lyophilized standards on a solid surface to ensure the pellet is at the bottom of the vial Reconstitute a single vial of standards with 781 ul of the appropriate diluent Optional at the same time reconstitute the controls vial with 250 ul of the appropriate diluent as summarized in Table 4 Controls do not require further dilution Vortex the reconstituted standards and controls at medium speed for 5 sec then incubate on ice for 30 min It is important that reconstitution of standards and controls is started and ended at the same time Be consistent with this incubation time to ensure optimal assay performance and reproducibility During the incubation period prepare the samples as instructed in the Prepare Samples section Prepare the Standard Dilution Series The following procedure produces an eight point standard curve with a threefold dilution between each point2. COOSSOCCCE COOSOCSCOSE Fig 4 Plate formatting 24 Enter a dilution factor of 4 and click Calculate The concentrations for each standard point will be populated for all analytes in the table Optional enter the lot number of the vial of standards into the Standard Lot box and click Save Click Enter Controls Info a For user specified controls select an analyte from the dropdown menu then enter a description and concentration Repeat for each additional analyte in the assay For the controls supplied with the 9 plex fixed panel only format the appropriate wells as controls enter descriptions but leave the concentrations blank Alternatively the controls can be formatted as samples with clear descriptions such as control high and control low In any case the expected control ranges provided are not entered into Bio Plex Manager software version 6 1 and earlier Click Enter Sample Info and enter sample information and the appropriate dilution factor Click Run Protocol and confirm that the assay settings are correct a Refer to Table 12 for the recommended RP1 PMT setting Protocols using alternative PMT settings should be validated by the end user Confirm that data acquisition is set to 50 beads per region In Advanced Settings confirm that the bead map is set to 100 region the sample size is set to 50 ul and the doublet discriminato
3. After the streptavidin PE incubation step slowly remove and discard the sealing tape 10 Wash the plate three times with 100 ul of wash buffer per well 11 To resuspend beads for plate reading add 125 ul assay buffer to each well Cover the plate with a new sheet of sealing tape Shake at room temperature at 850 50 rpm for 30 sec slowly remove and discard the sealing tape Ensure that the plate cover has been removed before placing the plate on the reader 21 Table 12 Read the plate using the appropriate instrument settings Instrument RP1 PMT DD Gates Bead Events Bio Plex 100 200 Low 5 000 low 25 000 high 50 Bio Plex 3D Standard Select MagPlex beads 50 Bio Plex MAGPIX N A use default instrument settings Or similar Luminex based system 9 Read Plate Bio Plex Manager software is recommended for all Bio Plex Pro assay data acquisition and analysis Instructions for Luminex xPONENT software are also included For instructions using other xMAP system software packages contact Bio Rad Technical Support or your regional Bio Rad field applications specialist Prepare Protocol in Bio Plex Manager Software Version 6 0 and Higher The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete A protocol file specifies the analytes in the assay the plate wells to be read sample information the values of standards and controls and instrument setti
4. Pipet carefully using calibrated pipets and use a new pipet tip for every volume transfer i Label eight 1 5 ml polypropylene tubes S2 through S8 and Blank Alternatively using Titertube micro test tubes may prove to be more convenient if a multichannel pipet will be used to load the plate Add 150 ul of the appropriate diluent to tubes S2 S8 Figure 3 Vortex reconstituted standards at medium speed for 5 sec before removing any volume Transfer 75 pl to the S2 tube containing the chosen standard diluent Vortex for 5 sec 13 4 Use anew pipet tip to transfer 75 ul from the S2 tube to the S3 tube Vortex for 5 sec 5 Continue with 1 3 threefold serial dilutions as shown in Figure 3 6 Use reconstituted and diluted standards and controls immediately Do not freeze for future use 75 75 75 75 75 75 75 Transfer Volume pl DXF RE XGRE NERNEY J Reconstituted Standard 150 150 150 150 150 150 150 150 Diluent pl si 2 3 s4 s5 s S7 s8 Blank Fig 3 Preparing a threefold dilution series with a single reconstituted standard 6 Prepare Samples General guidelines for preparing different sample types are provided here For more information consult publications listed in Bio Rad bulletin 5297 available for download at www bio rad com or contact Bio Rad Technical Support Please refer to the Important Considerations section for specific sample compatibility a
5. a o n 2 a o v R R 8 o 8 2 2 m R o R p 7 Run Protocol Fig 2 Suggested plate layout For detailed instructions on plate formatting in Bio Plex Manager see the Read Plate section 8 2 Prepare Instrument These directions are specific for the Bio Plex 100 200 reader To prepare either a Bio Plex 3D or Bio Plex MAGPIX reader consult their respective user manuals Note While the instrument is warming up bring the 10x wash buffer assay buffer and diluents to room temperature Keep other items on ice until needed Also begin to thaw frozen samples Start up and calibrate the Bio Plex system with Bio Plex Manager software prior to setting up the assay The calibration kit should be run daily or before each use of the instrument to standardize the fluorescent signal For instructions on using other xMAP system software packages contact Bio Rad Technical Support The validation kit should be run monthly to ensure optimal performance of fluidics and optics systems Refer to either the software manual or online Help for directions on how to conduct validation Start Up System Bio Plex 100 200 or similar 1 Empty the waste bottle and fill the sheath fluid bottle before starting if high throughput fluidics HTF are not present This will prevent fluidic system backup and potential data loss 2 Turn on th
6. Once thawed keep samples on ice Prepare dilutions just prior to the start of the assay and equilibrate to room temperature before use Prepare sample dilutions in microcentrifuge tubes Alternatively if a multichannel pipet will be used to load the plate aliquot the required volumes into Titertube micro test tubes a Do not freeze diluted samples 14 Table 5 Summary of recommended sample diluents and dilution factors Sample Type Dilution Factor Diluent Serum and plasma MMP 1 10 dilution Sample diluent HB Fluids MMP 1 4 and 1 40 Diluent 0 5 BSA w v Serum and Plasma Note If using plasma EDTA or citrate is preferred as an anticoagulant Heparin treated plasma while compatible with Bio Plex Pro assays may absorb certain soluble proteins of interest Avoid using hemolyzed samples as this may lead to false positive results 1 Draw whole blood into collection tubes containing anticoagulant Invert tubes several times to mix 2 For serum allow blood to clot at room temperature for 30 to 45 min For plasma proceed directly to the centrifugation steps 8 Perform centrifugation at 1 000 x g for 15 min at 4 C and transfer the serum or plasma to a clean polypropylene tube 4 To completely remove platelets and precipitates centrifuge again at 10 000 x g for 10 min at 4 C Alternatively filter the samples with a 0 8 0 2 um dual filter to prevent clogging 5 Dilute samples tenfold 1 10 by adding 1 volum
7. incubation steps and prior to reading Reader is clogged Incorrect needle height of the reader Low Signal or Poor Sensitivity Standards reconstituted incorrectly Detection antibody or streptavidin PE diluted incorrectly 31 Possible Solutions Check your calculations and be careful to add the correct volumes Vortex for 30 sec at medium speed before aliquoting beads Do not apply vacuum to the filter plate for longer than 10 sec after the buffer is completely drained from each well Shake the plate at 850 50 rom during incubation steps and for 30 sec immediately before reading the plate Refer to the troubleshooting guide in the Bio Plex System Hardware Instruction Manual bulletin 10005042 Adjust the needle height to coincide with the plate type provided in the kit Follow the standard preparation instructions carefully Check your calculations and be careful to add the correct volumes Possible Causes High Background Signal Incorrect buffer was used for example assay buffer used to dilute standards Accidentally spiked blank wells Detection antibodies or streptavidin PE incubated too long Poor Recovery Expired Bio Plex reagents were used Incorrect amounts of components were added Microplate shaker set to an incorrect speed High end saturation of the standard curve Controls do not fall within expected ranges Possible Solutions Use standard diluent HB or diluent simi
8. method of choice but do not perform the wash step 3 Add 100 ul of assay buffer to each well Cover the plate with a new sheet of sealing tape Shake the plate at 850 50 rpm for 30 sec Slowly remove the sealing tape before placing the plate on the plate reader 4 Repeat the Acquire Data steps to reacquire data The data acquired should be similar to those acquired initially however the acquisition time will be extended because the wells have fewer beads 26 Data Analysis Controls If the controls were run in the assay plate open the results rbx file click on Report Table and locate the control wells Visually compare the observed concentrations of the high and low controls in the Report Table against the lot specific control ranges shown in the product data sheet Note Expected control ranges are provided for reference and should be used as general guidelines Actual results may vary for some operators If the controls do not fall within the expected ranges please refer to the troubleshooting section for possible causes and solutions Removing Outliers Outliers are identified as standard data points that do not meet accuracy or precision requirements and should be considered invalid when performing curve fitting As such they should be removed to generate a more realistic and accurate standard curve This may result in an extended assay working range and allow quantitation of samples that might otherwise be considered
9. out of range In Bio Plex Manager software version 6 0 and higher outliers can be automatically removed by selecting the Optimize button in the Standard Curve window In Bio Plex Manager software 5 0 and earlier versions outliers also can be manually selected in the Report Table Visit online Help to learn more about the standard curve optimizer feature and how outliers are determined Previous Versions of Bio Plex Manager Software For instructions on using previous versions of Bio Plex Manager software please contact Bio Rad Technical Support 27 Luminex xPONENT Software Although guidelines are provided here consult the xPONENT software manual for more details Perform a system initialization with Luminex s calibration and performance verification kit as directed by Luminex Select Batches to set up the protocol and follow the information under Settings Note The instrument settings described below apply to Luminex 100 200 and FLEXMAP 8D or Bio Plex 3D instruments For the Bio Plex MAGPIX reader use the default instrument settings 1 Select MagPlex as the bead type for magnetic beads which automatically sets the DD gates 2 Volume 50 ul 8 Refer to Table 12 to select the appropriate PMT setting for your instrument 4 Plate name 96 well plate 5 Analysis type Quantitative 5PL Curve Fit 6 Number of standards 8 Select Analytes to set up the panel 1 Enter pg ml in the Units field 2 En
10. the wells to prevent leaking from the bottom 18 Add Coupled Beads Samples Standards Blank and Controls 1 Cover unused wells of the assay plate with sealing tape 2 Prewet the filter plate Skip this step if using a flat bottom plate Prewet the wells with 100 ul assay buffer and remove the liquid by vacuum filtration Dry the bottom of the filter plate thoroughly by blotting on a clean paper towel 3 Vortex the diluted 1x beads for 30 sec at medium speed Pour into a reagent reservoir and transfer 50 ul to each well of the assay plate Tip A multichannel pipet is highly recommended for ease of use and efficiency 4 Wash the plate two times with 100 pl Bio Plex wash buffer per well using the wash method of choice 5 Vortex the diluted samples standards blank and controls at medium speed for 5 sec Transfer 50 ul of each to the appropriate well of the assay plate changing the pipet tip after every volume transfer 6 Cover plate with a new sheet of sealing tape and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 1 hr at RT Note Be consistent with this incubation time and shaker setting for optimal assay performance and reproducibility Prepare and Add Detection Antibodies 1 While the samples are incubating use Tables 9 and 10 or the Calculation Worksheet on pages 35 36 to calculate the volume of detection antibodies and Bio Plex detection antibody diluent HB needed to prepar
11. ul 5 6 7 g Volume of assay buffer required ul ul ul 4 7 8 2 Determine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x x 25 ul ul 1 9 b Include 25 excess to ensure enough volume ul x 0 25 ul 9 10 c Total volume of 1x detection antibodies ul ul ul 9 10 11 d Enter the number of singleplex sets or analytes that will be multiplexed 5 e Volume of 20x detection antibodies required from each stock tube ul 20 ul 11 12 f Total volume of combined detection antibody stock ul x ul 12 5 13 g Volume of detection antibody diluent HB required ul ul ul 11 13 14 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 ul streptavidin PE 1x x 50 ul ul 1 15 b Include 25 excess to ensure enough volume ul x 0 25 ul 15 16 c Total volume of 1x streptavidin PE ul ul ul 15 16 17 d Volume of 100x streptavidin PE required ul 100 ul 17 18 e Volume of assay buffer required ul ul ul 17 18 19 36 Safety Considerations Eye protection and gloves are recommended when using these products Consult the MSDS for additional information Bio Plex Pro assays contain components of animal origin This material should be handled as if capable of transmitting infectious agents Use universal precautions These components should be handled at Biosafety Level 2 containment as defined by the U S government publication Biosafety in Microb
12. Bio Plex Pro Human MMP Assays Instruction Manual For technical support call your local Bio Rad office or in the U S call 1 800 424 6723 For research use only Not for diagnostic procedures Table of Contents Introduction Principle Kit Contents and Storage Recommended Materials Assay Workflow Important Considerations Detailed Instructions 1 Plan Plate Layout Prepare Instrument Prepare Wash Method Preparing Buffer and Diluent Prepare Standards and Controls Prepare Samples Prepare Coupled Beads ONO a FF W DY Run Assay 9 Read Plate Troubleshooting Guide Plate Layout Template Calculation Worksheet Safety Considerations Legal Notices Ordering Information oAnN NO oO A N yo i N OOD FN O U oOo U WO wb ND N N A A O Introduction The matrix metalloproteinases MMPs are a family of 24 zinc proteases with essential roles in breaking down components of the extracellular matrix ECM MMPs play important roles in tissue remodeling associated with various physiological and pathological processes such as morphogenesis angiogenesis tissue repair cirrhosis arthritis and metastasis MMPs substrates include ECM components cytokines chemokines growth factors and binding proteins cell cell adhesion molecules and other proteinases The MMPs are inhibited by specific endogenous tissue inhibitors of metalloproteinases TIMPs Multiplexing with Bio Plex Pro MMP Assa
13. ate Layout Determine the total number of wells in the experiment using the Plate Layout Template on page 34 or the Plate Formatting tab in Bio Plex Manager A suggested plate layout is shown in Figure 2 with all conditions in duplicate I Assign standards to columns 1 and 2 with the highest concentration in row A and the lowest concentration in row H 2 Assign the blank to wells A3 and A4 The blank should consist of your chosen standard diluent HB Note that Bio Plex Manager automatically subtracts the blank B MFI value from all other assay wells 8 User specified controls as well as the controls supplied in premixed kits are assigned to wells in columns 3 and 4 4 The remainder of the plate is available for samples 5 Once the total number of wells is known you can calculate the required volumes of beads detection antibody and streptavidin PE Use Tables 6 7 9 10 and 11 respectively or the Calculation Worksheet on pages 35 36 Elia 2 a Oke Legend Protocol Settings 2 Select Analytes srr K ADO amp B OG 1 8 4 Enter Standards info F OG 5 Enter Controls Info H 6 Enter Sample Info Plate Formatting Plate Groupings A T2 ESA v G 0O DW PD s Standard 18 18 19 19 20 20 Blank 21 21 22 22 23 23 3 ce 2 OO F E O Samples i TOO Controls a a
14. ate the volume of coupled beads and assay buffer needed to prepare a 1x stock Add the required volume of Bio Plex assay buffer to a 15 ml polypropylene tube 16 3 Vortex the 20x stock of coupled beads at medium speed for 30 sec Carefully open the cap and pipet any liquid trapped in the cap back into the tube This is important to ensure maximum bead recovery Do not centrifuge the vial doing so will cause the beads to pellet 4 Dilute coupled beads to 1x by pipetting the required volume into the 15 ml tube Vortex Each well of the assay requires 2 5 ul of the 20x stock adjusted to a final volume of 50 ul in assay buffer 5 Protect the beads from light with aluminum foil Equilibrate to room temperature prior to use Note To minimize volume loss use a 200 300 ul capacity pipet to remove beads from the 20x stock tube If necessary perform the volume transfer in two steps Do not use a 1 000 ul capacity pipet and or wide bore pipet tip Preparing 1x coupled beads from 20x stock includes 20 excess volume Table 6 Premixed panel or one singleplex assay of Wells 20x Beads ul Assay Buffer pl Total Volume pl 96 288 5 472 5 760 48 144 2 736 2 880 Table 7 Mixing singleplex assays 20x Beads pl 20x Beads ul of Wells Singleplex 1 Singleplex 2 Assay Buffer pl Total Volume pl 96 288 288 5 184 5 760 48 144 144 2 592 2 880 17 8 Run Assay Considerations a Bring all assay components an
15. contamination of standards is suspected check the standard replicate value and be careful when adding samples to the wells Matrix effects could also produce negative sample values Bio Plex Manager software automatically subtracts the Blank B MFI value from all other assay wells While this has no impact on observed concentrations of samples within the assay working range it may result in a negative MFI value if the Blank s MFI value is greater than either the standard or the sample value If this is undesirable then reformat the blank wells as Sample X or Control C in the protocol or results file Check if any interfering components additives or gel from separators were introduced into the samples Avoid using hemolyzed and heavily lipemic samples Remove visible particulate in samples by centrifugation Avoid multiple freeze thaw cycles of samples 33 Plate Layout Template cL LL OF m Oo A WU A lt 34 Calculation Worksheet If using either a premixed panel or one singleplex assay follow these directions Plan the plate layout and enter the number of wells to be used in the assay 1 Determine the volume of 1x coupled beads needed a Each well requires 50 ul of coupled beads 1x x 50 pl ul 2 b Include 20 excess to ensure enough volume ul x 0 20 ul 2 3 c Total volume of 1x coupled beads ul ul ul 2 3 4 d Volume of 20x coupled beads required
16. d diluent HB to 6 ml sample diluent HB 11 5 Prepare Standards and Controls General Instructions It is essential to prepare standards and controls if included exactly as described in this section Incorrect preparation may lead to low signal or variable measurements from plate to plate a The product data sheet provided with the standards lists the most concentrated point on the standard curve S1 Enter this information into Bio Plex Manager software as instructed in section 9 Using the Controls optional A single vial of controls is provided with the 9 plex fixed panel only Their use is intended for monitoring the day to day quality of assay results Selecting a Diluent for Standards and Controls Refer to Table 4 for recommended diluents based on different sample types In order to meet the lot specific control ranges provided on the product data sheet both the standards and controls should be reconstituted in Bio Plex standard diluent HB If reconstituting in a different diluent users will need to establish validate their own control ranges or acceptance criteria Table 4 Summary of recommended diluents for standards and controls Diluent for Standards Sample Type and Controls Add BSA Serum and plasma 10 serum based standard diluent None Culture media with serum Culture medium None Culture media serum free Culture medium To 0 5 final Lavage sputum other fluids Sample diluent HB To 0 5 final
17. d samples to room temperature before use Use calibrated pipets and pipet carefully avoiding bubbles Pay close attention to vortexing shaking and incubation instructions Deviation from the protocol may result in low assay signal and assay variability a Assay incubations are carried out on a shaker at 850 50 rpm at room temperature RT Cover the plate with sealing tape and protect from light with aluminum foil Table 8 Summary of wash options and protocols After each assay step select the appropriate Bio Plex Pro wash station program or perform the appropriate manual wash step as summarized below Handheld Magnet or Bio Plex Pro Wash Station Vacuum Manifold Assay Step Magnetic Program Manual Wash Steps Add beads to plate MAG x2 2x 100 ul Sample incubation Detection Ab incubation MAG x3 3 x 100 ul SA PE incubation Considerations When Using a Vacuum Manifold a After each incubation place the filter plate on a calibrated vacuum apparatus and remove the liquid by vacuum filtration To wash add 100 ul wash buffer to each well and remove the liquid as before Ensure that all wells are exposed to the vacuum a Thoroughly blot the bottom of the filter plate with a clean paper towel between each vacuum step to prevent cross contamination a Place the assay plate on the plastic plate holder tray as needed Before each incubation gently cover the plate with a new sheet of sealing tape Avoid pressing down on
18. e a 1x stock Detection antibodies should be prepared 10 min before use 2 Add the required volume of Bio Plex detection antibody diluent HB to a 15 ml polypropylene tube 3 Vortex the 20x stock of detection antibodies for 15 20 sec at medium speed then perform a 30 sec spin to collect the entire volume at the bottom of the tube 19 Dilute detection antibodies to 1x by pipetting the required volume into the 15 ml tube Vortex Each well of the assay requires 1 25 ul of the 20x stock adjusted to a final volume of 25 ul in detection antibody diluent HB Preparing 1x detection antibodies from 20x stock includes 25 excess volume Table 9 Premixed panel or one singleplex assay 20x Detection Detection Antibody of Wells Antibodies pl Diluent pl Total Volume pl 96 150 2 850 3 000 48 75 1 425 1 500 Table 10 Mixing singleplex assays 20x Detection 20x Detection Detection Antibodies yl Antibodies pl Antibody of Wells Singleplex 1 Singleplex 2 Diluent pl Total Volume ul 96 150 150 2 700 3 000 48 75 75 1 350 1 500 After incubating the beads samples standards blank and controls slowly remove and discard the sealing tape Wash the plate three times with 100 ul wash buffer per well Vortex the diluted 1x detection antibodies at medium speed for 5 sec Pour into a reagent reservoir and transfer 25 ul to each well of the assay plate using a multichannel pipet Cover plate with a new sheet
19. e of sample to 9 volumes of Bio Plex sample diluent HB for example 15 ul sample 135 pl sample diluent HB 6 Assay samples immediately or aliquot into single use tubes and store at 70 C Avoid repeated freeze thaw cycles 15 Cell Culture Supernatant I Collect supernatants and centrifuge at 1 000 x g for 15 min at 4 C For cell lines cultured in serum free culture media collect samples and add BSA as a carrier protein to a final concentration of at least 0 5 to stabilize protein analytes and to prevent adsorption to labware Transfer to a clean polypropylene tube If cellular debris or precipitates are present centrifuge again at 10 000 x g for 10 min at 4 C We recommend testing undiluted samples first If high levels of analyte are expected samples can be further diluted in culture medium Rarely would samples need to be diluted greater than 1 10 Assay immediately or store samples in single use aliquots at 70 C Avoid repeated freeze thaw cycles Lavage Sputum and Other Biological Fluid Samples Keep all samples on ice until ready for use The appropriate sample dilution factor should be optimized by the user 1 If required dilute the sample in Bio Plex sample diluent HB with BSA added to a final concentration of 0 5 Centrifugation at 10 000 x g for 10 min at 4 C may be required to clarify the sample Prepare Coupled Beads Use Tables 6 7 or the Calculation Worksheet on pages 35 36 to calcul
20. e reader XY platform and HTF if included Allow the system to warm up for 30 min if not already done 3 Select Start up ny and follow the instructions If the system is idle for 4 hr without acquiring data the lasers will automatically turn off To reset the 4 hr countdown select Warm up and wait for the lasers optics to reach operational temperature Calibrate System 1 Select Calibrate Oo and confirm that the default values for CAL1 and CAL2 are the same as the values printed on the bottle of Bio Plex calibration beads Use the Bio Plex system low RP1 target value 2 Select OK and follow the software prompts for step by step instructions for CAL1 and CAL2 calibration Note In Bio Plex Manager version 6 1 and higher startup warm up and calibration can be performed together by selecting the Start up and calibrate icon X 3 Prepare Wash Method Bio Plex Pro assays are compatible with both magnetic separation and vacuum filtration methods However for best results we recommend performing the assays in a flat bottom plate with magnetic separation Table 3 Summary of compatible wash stations and plate types Wash Method Wash Station Assay Plate Magnetic separation Bio Plex Pro Flat bottom plate Bio Plex handheld magnetic washer Vacuum filtration Vacuum manifold manual Filter plate Setting Up the Bio Plex Pro Wash Station The wash station should be primed before use For more informat
21. ed in MMP Express kits Other Components and Accessories Bio Plex software wash buffer Bio Plex Pro flat bottom plates and streptavidin PE are also available individually For more information go to www bio rad com bio plex 38 Life Science Group 10041639 Rev A Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 3065 7550 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 6965 Germany 089 31 8840 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 1404 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026 674 55 05 Taiwan 886 2 2578 7189 Thailand 1800 88 22 88 United Kingdom 020 8328 2000 Sig 1213
22. edian fluorescence intensity MFI as well as concentration pg ml The concentration of analyte bound to each bead is proportional to the MFI of reporter signal Using Bio Plex Data Pro software data from multiple instrument runs can be combined into a single project for easy data management quick visualization of results and simple statistical analysis Kit Contents and Storage Reagents Supplied Bio Plex Pro human MMP assays are available in a convenient all in one kit format that includes assay reagent and diluent components in a single box Table 1 Contents of 1 x 96 well kits Component Quantity Coupled magnetic beads 20x 1 tube Detection antibodies 20x 1 tube Standards 1 vial Control 1 vial Sample diluent HB 1 bottle 80 ml Detection antibody diluent HB 1 bottle 3 5 ml Standard diluent HB 1 bottle 10 ml Assay buffer 1 bottle 60 ml Wash buffer 10x 1 bottle 60 ml Streptavidin PE 100x 1 tube Assay plate 96 well flat bottom plate 1 plate Sealing tape 1 pack of 4 Assay quick guide 1 booklet Product data sheet 1 sheet Provided with the 9 plex fixed panel only Volumes shown are approximate Storage and Stability Kit contents should be stored at 4 C and never frozen Coupled magnetic beads and streptavidin PE should be stored in the dark All components are guaranteed for a minimum of six months from the date of purchase when stored as specified Table 2 Recommended mate
23. iological and Biomedical Laboratories Centers for Disease Control 1999 Legal Notices Acrodisc and Supor are trademarks of Pall Corporation MagPlex xMAP xPONENT FLEXMAP 3D MAGPIX and Luminex are trademarks of Luminex Corporation The Bio Plex suspension array system includes fluorescently labeled microspheres and instrumentation licensed to Bio Rad Laboratories Inc by the Luminex Corporation 37 Ordering Information Detailed ordering information can be found at www bio rad com bio plex Catalog Premixed Multiplex Assay Panel 171 AMO01M Bio Plex Pro Human MMP Panel 9 Plex 1 x 96 well Singleplex Set Various Bio Plex Pro Human MMP Singleplex Sets 1 x 96 well requires reagent kit Ill and MMP standards to run an assay Individual Components and Accessories 171 304090 Bio Plex Pro Reagent Kit III with Filter Plate 1 x 96 well 171 304090M Bio Plex Pro Reagent Kit Ill with Flat Bottom Plate 1 x 96 well 171 DMo001 Bio Plex Pro Human MMP Standard pkg of 1 vial 171 DM0501 Bio Plex Pro Human MMP Standard pkg of 50 vials 171 304502 Filter Plate 1 x 96 well with clear plastic lid and tray Bio Plex Express Assays You Mix Fast and economical custom assay service using the Bio Plex Assay Builder www bio rad com bio plex assaybuilder to select analytes and plate type of interest Assays are supplied as individual sets of coupled beads and detection antibodies in the all in one kit format Controls are not includ
24. ion refer to the Bio Plex Pro Wash Stations Quick Guide bulletin 5826 1 Install the appropriate plate carrier on the wash station 2 Use the Prime procedure to prime channel 1 with 1x wash buffer 10 Setting Up the Bio Plex Handheld Magnetic Washer Place an empty flat bottom plate on the magnetic washer by sliding it under the retaining clips Push the clips inward to secure the plate Make sure the plate is held securely If needed the clips can be adjusted for height and tension For detailed instructions refer to the user guide bulletin 10023087 Setting Up a Vacuum Manifold Calibrate the vacuum manifold by placing a standard 96 well flat bottom plate on the unit and adjusting the pressure to 1 to 3 Hg In general 100 ul liquid should take 3 4 sec to clear the well For more detailed instructions refer to bulletin 10005042 4 Preparing Buffer and Diluent Prepare Wash Buffer 1 Bring the 10x stock solution to room temperature 2 If crystals are present ensure that they are completely dissolved Mix the 10x stock solution by inversion before preparing the 1x wash buffer 3 To prepare 1x wash buffer dilute 1 part 10x stock solution with 9 parts deionized water Prepare 10 Serum Based Standard Diluent 1 Bring the standard diluent HB to room temperature 2 To prepare 10 serum based standard diluent dilute 1 part standard diluent HB with 1 5 parts sample diluent HB for example add 4 ml standar
25. lar to final sample matrix to dilute standards Do not add any antigens to the blank wells Follow the procedure incubation time precisely Check that reagents have not expired Use new or nonexpired components Check your calculations and be careful to add the correct volumes Check the microplate shaker speed and use the recommended setting Setting the speed too high may cause splashing and contamination Use the recommended plate shaker Setting the speed too low may cause low assay signal and false plateau or high end saturation of standard curves Make sure that correct shaker soeed and incubation times are used Remove S1 from data analysis if needed Make sure that the controls are reconstituted at the same time as standards and in the same diuent Standard diluent HB Incubate for precisely 30 min 32 Possible Causes Poor Recovery Improper pipetting technique Impact of Sample Matrix Negative MFI values in samples or standards Poor precision in serum and plasma sample measurements Possible Solutions Pipet carefully when adding standards samples detection antibodies and streptavidin PE especially when using a multichannel pipet Use a calibrated pipet Change pipet tip after every volume transfer If samples contain little or no analyte negative values observed may be due to statistical variation If assay drift is suspected retest the samples by positioning them next to the standards If
26. log 170 20100 Bio Rad catalog 171 025001 Bio Rad catalog 223 9390 IKA catalog 320 8000 VWR catalog 57019 600 Bio Rad catalog 732 6470 Bio Rad catalog 166 0610 VistaLab catalog 3054 1002 VistaLab catalog 3054 1004 VistaLab catalog 3054 1006 Pall Life Sciences catalog 4187 Bio Rad catalog 171 304502 Bio Rad catalog 171 051555 Other 15 ml polypropylene tubes for reagent dilutions calibrated pipets pipet tips sterile distilled water aluminum foil absorbent paper towels 1 5 or 2 ml microcentrifuge tubes and standard flat bottom microplate for calibrating vacuum manifold Assay Workflow Prewet wells for filter plate only Add 50 ul 1x beads to wells Wash 2 x 100 ul Add 50 ul standards samples controls incubate on shaker at 850 rpm for 1 hr at RT Wash 3 x 100 ul Add 25 pl 1x detection antibody incubate on shaker at 850 rpm for 30 min at RT Wash 3 x 100 ul Add 50 ul 1x streptavidin PE incubate on shaker at 850 rpm for 10 min at RT Wash 3 x 100 ul Resuspend in 125 ul assay buffer shake at 850 rpm for 30 sec Acquire data on Bio Plex system Important Considerations Instruments and Software The Bio Plex Pro assays described in this manual are compatible with all currently available Luminex based life science research instruments Assays can be read and analyzed with either Bio Plex Manager software or Luminex xPONENT software see the Run Assay section A
27. me Table 13 Bead regions for the human MMP panel Analyte Bead Region Analyte Bead Region Analyte Bead Region MMP 1 43 MMP 7 66 MMP 10 62 MMP 2 26 MMP 8 37 MMP 12 53 MMP 3 45 MMP 9 55 MMP 13 47 23 Note Do not use preset panels found in Bio Plex Manager software version 5 0 or earlier as the bead regions are not up to date 3 Click Format Plate and format the plate according to the plate layout created in section 1 Plan Plate Layout To modify the plate layout follow the steps below see Figure 4 a Select the Plate Formatting tab b Select the standards icon and drag the cursor over all the wells that contain standards Repeat this process for Blanks Controls c and Samples X Note that Bio Plex Manager automatically subtracts the blank MFI value from all other assay wells 4 Click Enter Standards Info in the Protocol Settings bar a Enter the highest concentration of each analyte in the top row labeled S1 of the table S1 concentration information is included with each vial of standards WE 2 a Ske Protocol Settings Plate Formatting Plate Groupings V O Ge a 1 SOLIE OOL Te 1 8 8 18 18 19 19 20 20 1 9 9 21 21 22 22 23 23 24 24 25 25 26 26 28 28 29 29 v c9 N A N A o 7 moO J gt
28. ngs Bio Plex Manager software versions 6 0 and higher contain protocols for most Bio Plex assays Choose from available protocols or create a new protocol To create a new protocol select File then New from the main menu Locate and follow the steps under Protocol Settings 1 Click Describe Protocol and enter information about the assay optional 22 2 Click Select Analytes and create a new panel Visually confirm the selected analytes and proceed to step 3 a Click Add Panel iss in the Select Analytes toolbar Enter a new panel name Select Bio Plex Pro Assay Magnetic from the assay dropdown list If using Bio Plex Manager version 5 0 or lower select MagPlex from the assay dropdown list b Click Add Enter the bead region number and name for the first analyte Click Add Continue to repeat for each analyte in the assay Refer to the bead regions in parentheses listed on the peel off label provided with the standards For reference bead regions are shown in Table 13 c Click Add when the last analyte has been added and click OK to save the new panel d Highlight analytes from the Available list left and move to the Selected list right using the Add button To move all analytes at once simply click Add All e lf some of the analytes need to be removed from the Selected list highlight them and select Remove If desired it is possible to rename the panel by clicking Rename Panel and entering a new panel na
29. of sealing tape and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 30 min at RT Prepare and Add Streptavidin PE SA PE While detection antibodies are incubating use Table 11 or the Calculation Worksheet on page 36 to calculate the volume of SA PE and assay buffer needed to prepare a 1x stock SA PE should be prepared 10 min before use 20 2 Add the required volume of assay buffer to a 15 ml polypropylene tube 3 Vortex the 100x stock of SA PE for 5 sec at medium speed Perform a 30 sec spin to collect the entire volume at the bottom of the tube 4 Dilute SA PE to 1x by pipetting the required volume into the 15 ml tube Vortex and protect from light until ready to use Each well of the assay requires 0 5 ul of the 100x stock adjusted to a final volume of 50 ul in assay buffer Table 11 Preparing 1x SA PE from 100x stock includes 25 excess volume of Wells 100x SA PE ul Assay Buffer pl Total Volume pl 96 60 5 940 6 000 48 30 2 970 3 000 5 After detection antibody incubation slowly remove and discard the sealing tape 6 Wash the plate three times with 100 ul of wash buffer per well Vortex the diluted 1x SA PE at medium speed for 5 sec Pour into a reagent reservoir and transfer 50 ul to each well using a multichannel pipet 8 Cover plate with a new sheet of sealing tape and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 10 min at RT 9
30. ossible Causes High Intra Assay CV Improper pipetting technique Reagents and assay components not equilibrated to room temperature prior to pipetting Contamination with wash buffer during wash steps Slow pipetting of samples and reagents across the plate Bio Plex wash station insufficient washing due to clogged pins Possible Solutions Pipet carefully when adding standards controls samples detection antibodies and streptavidin PE especially when using a multichannel pipet Use a calibrated pipet Change pipet tip after every volume transfer All reagents and assay components should be equilibrated to room temperature prior to pipetting During the wash steps be careful not to splash wash buffer from one well to another Be sure that the wells are filtered completely and that no residual volume remains Ensure that the microplate shaker setting is not too high Reduce the microplate shaker speed to minimize splashing Sample pipetting across the entire plate should take less than 4 min Reagent pipetting across the entire plate should take less than 1 min Clean dispensing pins with the thicker of the two cleaning needles provided with washer Perform regular rinses to minimize salt buildup 30 Possible Causes Low Bead Count Miscalculation of bead dilution Beads clumped in multiplex bead stock tube Vacuum on for too long when aspirating buffer from wells Assay plate not shaken enough during
31. r DD gates are set to 5 000 Low and 25 000 High In Bio Plex Manager software versions 4 0 4 1 4 1 1 and 5 0 check Override Gates and set the DD gate values as indicated Select Start name and save the rbx file and begin data acquisition The Run Protocol pop up screen will appear Click Eject Retract to eject the plate carrier 25 Acquire Data 1 Shake the assay plate at 850 50 rpm for 30 sec and visually inspect the plate to ensure that the assay wells are filled with buffer Slowly remove the sealing tape and any plate cover before placing the plate on the plate carrier 2 Click Run Protocol and on the pop up screen select Load Plate and click OK to start acquiring data 3 Use the Wash Between Plates command after every plate run to reduce the possibility of clogging the instrument 4 If acquiring data from more than one plate empty the waste bottle and refill the sheath bottle after each plate if HTF are not present Select Wash Between Plates and follow the instructions Then repeat the Prepare Protocol and Acquire Data instructions 5 When data acquisition is complete select Shut Down and follow the instructions Reacquire Data It is possible to acquire data from a well or plate a second time using the Rerun Recovery mode located below Start in the Run Protocol step Any previous data will be overwritten 1 Check the wells from which data will be reacquired 2 Aspirate the buffer with the wash
32. rials Item Ordering Information Bio Plex Pro MMP Assays Quick Guide Bio Plex MAGPIX 200 system or Luminex system with HTF Bio Plex validation kit Note Run the validation kit monthly to ensure optimal performance of fluidics and optics systems Bio Plex calibration kit Note Run the calibration kit daily to standardize fluorescence signal Bio Plex Pro wash station For use with magnetic bead based assays only Bio Plex handheld magnetic washer For use with magnetic bead based assays only Bio Plex Pro flat bottom plates 40 x 96 well For magnetic separation on the Bio Plex Pro wash station Titertube micro test tubes For preparing replicate standards samples and controls prior to loading the plate Microtiter plate shaker IKA MTS 2 4 shaker for 2 or 4 microplates or Barnstead Lab Line Model 4625 plate shaker or equivalent capable of 300 1 100 rpm Aurum vacuum manifold For vacuum filtration BR 2000 vortexer Reagent reservoirs 25 ml For capture beads and detection antibodies Reagent reservoir 50 ml for reagents and buffers Pall Life Science Acrodisc 25 mm PF syringe filter 0 8 0 2 um Supor membrane Filter plate 1 x 96 with clear plastic lid and tray Bio Plex Manager MP Software Upgrade Bulletin 10041637 download at www bio rad com bio plex Bio Rad catalog 171 000205 Bio Rad catalog 171 203001 Bio Rad catalog 171 203060 Bio Rad catalog 300 34376 Bio Rad cata
33. ssay Procedures Please pay close attention to vortexing shaking and incubation times and to Bio Plex reader PMT RP1 setting as these have been optimized specifically for each assay panel Assay Quick Guide Each assay kit comes complete with a printed Bio Plex Pro Assay Quick Guide bulletin 10041637 which can be used to prepare and run a full 1 x 96 well assay plate Users can also download a copy at www bio rad com bio plex Bead Regions and Multiplexing Compatibility a Bead regions for all analytes are listed in the Read Plate section Do not mix analytes between different Bio Plex panels or reagent kits Resulting standard curves and sample values may be inaccurate There is a small chance of cross reactivity caused by MMP 13 in EDTA plasma from cancer patients In a small study N 17 of breast and colon cancer samples MMP 13 showed cross reactivity in four EDTA plasma samples 24 and none in the matching serum samples Therefore it is recommended not to mix MMP 13 with any other assays when testing EDTA plasma cancer samples Use only one type of matrix in each study to ensure data continuity placing emphasis on consistency in sample collection preparation and storage Detailed Instructions The following pages provide detailed instructions for each step of the assay procedure including preparation running the assay and reading the plate with Bio Plex Manager and Luminex xPONENT software 7 1 Plan Pl
34. tal signal processor that efficiently manages the fluorescence data Assay Format Bio Plex Pro assays are essentially immunoassays formatted on magnetic beads The assay principle is similar to that of a sandwich ELISA Figure 1 Capture antibodies directed against the desired biomarker are covalently coupled to the beads Coupled beads react with the sample containing the biomarker of interest After a series of washes to remove unbound protein a biotinylated detection antibody is added to create a sandwich complex The final detection complex is formed with the addition of streptavidin phycoerythrin SA PE conjugate Phycoerythrin serves as a fluorescent indicator or reporter Biomarker of fig Streptavidin x lt i gt xk MS Bead vache eae Fluorescent Capture Biotinylated Reporter Antibody Detection Antibody Fig 1 Bio Plex sandwich immunoassay Data Acquisition and Analysis Data from the reactions are acquired using a Bio Plex system or similar Luminex based reader When a multiplex assay suspension is drawn into the Bio Plex 200 reader for example a red 635 nm laser illuminates the fluorescent dyes within each bead to provide bead classification and thus assay identification At the same time a green 632 nm laser excites PE to generate a reporter signal which is detected by a photomultiplier tube PMT A high speed digital processor manages data output and Bio Plex Manager software presents data as m
35. ter 50 in the Count field 8 Select the bead region and enter the analyte name 4 Click Apply all for Units and Count Select Stds and Cirls 1 Enter standard concentrations lot number dilution factor and other information as applicable After the assay is complete select Results then select Saved Batches 28 Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio Plex Pro assays If you experience any of the problems listed below review the possible causes and solutions provided Poor assay performance may also be due to the Bio Plex suspension array reader To eliminate this possibility use the validation kit to determine whether the array reader is functioning properly Possible Causes High Inter Assay CV Standards and controls were not reconstituted consistently between assays Reconstituted standards controls and diluted samples were not stored properly Bottom of filter plate not dry 29 Possible Solutions Incubate the reconstituted standards for 30 min on ice Always be consistent with the incubation time and temperature Reconstituted standards and diluted samples should be prepared on ice as instructed Prior to plating the reconstituted standards and diluted samples should be equilibrated to room temperature Dry the bottom of the filter plate with absorbent paper towel preferably lint free to prevent cross well contamination P
36. ul 20 ul 4 5 e Volume of assay buffer required ul ul ul 4 5 6 2 Determine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x x 25 l ul 1 7 b Include 25 excess to ensure enough volume ul x 0 25 ul 7 8 c Total volume of 1x detection antibodies ul ul ul 7 8 9 d Volume of 20x detection antibodies required ul 20 ul 9 10 e Volume of detection antibody diluent required ul ul ul 9 10 11 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 ul streptavidin PE 1x x 50 ul ul 1 12 b Include 25 excess to ensure enough volume ul x 0 25 ul 12 13 c Total volume of 1x streptavidin PE ul ul ul 12 13 14 d Volume of 100x streptavidin PE required ul 100 ul 14 15 e Volume of assay buffer required ul ul ul 14 15 16 35 If mixing singleplex assays follow these directions Enter the number of wells to be used in the assay 1 1 Determine the volume of 1x coupled beads needed a Each well requires 50 ul coupled beads 1x x 50 ul ul 1 2 b Include 20 excess to ensure enough volume ul x 0 20 ul 2 3 c Total volume of 1x coupled beads ul ul ul 2 3 4 d Enter the number of singleplex sets or analytes that will be multiplexed 5 e Volume of 20x coupled beads required from each stock tube ul 20 ul 4 6 f Total volume of combined bead stocks xX ul
37. ys Bio Plex Pro MMP assays enable researchers to quantify multiple protein biomarkers in a single well of a 96 well plate in just 3 4 hours These robust immunoassays require as little as 5 ul of serum or plasma or 50 ul of other biological fluid The use of magnetic MagPlex beads allows researchers to automate wash steps on a Bio Plex Pro or similar wash station Magnetic separation offers greater convenience and reproducibility compared to vacuum filtration For more information please visit www bio rad com bio plex Principle Technology The Bio Plex multiplex system is built upon the three core elements of xMAP technology Fluorescently dyed magnetic microspheres also called beads each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension This allows simultaneous detection of up to 500 different molecules in a single well of a 96 well microplate on the Bio Plex 3D system up to 100 different molecules on the Bio Plex 200 system and up to 50 different molecules on the Bio Plex MAGPIX system a A dedicated plate reader The Bio Plex 200 and Bio Plex 3D systems are flow cytometry based instruments with two lasers and associated optics to measure the different molecules bound to the surface of the beads In the Bio Plex MAGPIX system the sample is injected into a chamber where the beads are imaged using LED and CCD technology a A high speed digi
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