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1. Perform centrifugation at 1 000 x g for 15 min at 4 C and transfer the serum or plasma to a clean polypropylene tube To completely remove platelets and precipitates centrifuge again at 10 000 x g for 10 min at 4 C Activate samples as described below and assay immediately or store untreated nonactivated samples in single use aliquots at 70 C Avoid repeated freeze thaw cycles 15 Cell Culture Supernatant 1 Collect supernatants and perform centrifugation 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 Activate samples as described below and assay immediately or store untreated nonactivated 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 determined by the user 1 Centrifuge at 10 000 x g for 10 min at 4 C to clarify the sample 2 Activate a portion of the sample as described below and if needed dilute in Bio Plex sample diluent with BSA added to a final concentration of at least 0 5 Lysates Users will2. 1 Plan Plate Layout Prior to running the assay determine the total number of wells in the experiment using the Plate Layout Template on page 36 or the Plate Formatting tab in Bio Plex Manager software A suggested plate layout is shown in Figure 2 with all conditions in duplicate 1 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 and be processed in the same manner as sample and standard wells Note that Bio Plex Manager automatically subtracts the blank B MFI value from all other assay wells 8 User defined controls 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 calculate the required volumes of beads detection antibody and streptavidin PE needed Use Tables 7 8 10 11 and 12 respectively or the Calculation Worksheet on pages 37 38 eB Fes OhH Legend Protocol Settings Fate Fomatine a Mig 2b a 5G ea 8S EE inet s Standard A OOSA 6 6 14 14 22 22 30 30 B 2 G7 Iz Essl le c OO 2 2 8 8 18 16 24 24 32 32 Blank o OOL J lle ILe 7 7 25 25 5 38 e OOl 10 10 18 18 26 26 24 4 x Samples E OGO 3 3 11 11 19 19
3. 21 2 Add the required volume of Bio Plex detection antibody diluent to a 15 ml polypropylene tube 3 Vortex the stock 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 4 Dilute detection antibodies to 1x by pipetting the required volume into the 15 ml tube Each well of the assay requires 1 25 ul 20x stock adjusted to a final volume of 25 ul in detection antibody diluent Preparing 1x detection antibodies from 20x stock includes 25 excess volume Table 10 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 11 Mixing singleplex assays 20x Detection 20x Detection Detection Antibodies yl Antibodies pl Antibody of Wells Singleplex 1 Singleplex 2 Diluent pl Total Volume pl 96 150 150 2 700 3 000 48 75 75 1 350 1 500 5 After incubating the beads samples standards and blank slowly remove and discard the sealing tape 6 Wash the plate three times with 100 pl wash buffer 7 Vortex the diluted 1x detection antibodies gently for 5 sec Pour into a reagent reservoir and transfer 25 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 1 hr at room temperature 22
4. All components are guaranteed for a minimum of 6 months from the date of purchase when stored as specified Reagents Required but Not Supplied TGF B is secreted as part of a complex that causes it to be inactive It is necessary to expose samples to acidic conditions in order to activate TGF B The following reagents are required a 1 N hydrochloric acid To make 100 ml of 1 N HCl add 8 33 ml of 12 N HCl slowly to 91 67 ml of deionized water and mix well a 1 2 N sodium hydroxide 0 5 M HEPES To make 100 ml of 1 2 NaOH 0 5 M HEPES add 12 ml of 10 N NaOH to 75 ml of deionized water and mix well Add 11 9 g of HEPES free acid MW 238 3 mix well and bring the final volume to 100 ml with deionized water Table 2 Recommended materials Item Ordering Information Bio Plex Pro Assays Quick Guide 5 Bio Plex 200 system or Luminex system with HTF Bio Plex validation kit Run the validation kit monthly to ensure optimal performance of fluidics and optics systems Bio Plex calibration kit Run the calibration kit daily to standardize fluorescence signal Bio Plex Pro wash station For use with magnetic bead based assays only Bio Plex Pro II wash station For use with both polystyrene nonmagnetic and magnetic bead based assays 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 Microtite
5. 27 27 35 35 Ss DOLL zl 20 2s 2 es 28 R H 55 13 13 21 21 29 29 37 37 e Gontrols Fig 2 Suggested plate layout For detailed instructions on plate formatting in Bio Plex Manager see section 8 Read Plate 9 2 Prepare Instrument Start up and calibrate the Bio Plex 100 200 or similar 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 To prepare either a Bio Plex 3D or Bio Plex MAGPIX reader consult its respective user manual The validation kit should be run monthly to ensure 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 the reader XY platform and HTF if included Allow the system to warm up for 30 min if not already done 3 Select Start up he 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
6. 3 plex Panel 1 x 96 171 W4001M TGF f Singleplex Sets Catalog Bio Plex Pro TGF f1 Set 1 x 96 171 V4001M Bio Plex Pro TGF f2 Set 1 x 96 171 V4002M Bio Plex Pro TGF f3 Set 1 x 96 171 V4003M Premixed Cytokine Panels Catalog Bio Plex Pro Human Cytokine 8 Plex Panel 1 x 96 M50 000007A Bio Plex Pro Human Cytokine 17 Plex Panel 1 x 96 M50 00031YV Bio Plex Pro Human Cytokine 21 Plex Panel 1 x 96 MFO OO5KMII Bio Plex Pro Human Cytokine 27 Plex Panel 1 x 96 M50 OKCAFOY Bio Plex Pro Human Cytokine Th1 Th2 Panel 1 x 96 MS50 00005L3 Bio Plex Pro Mouse Cytokine 8 Plex Panel 1 x 96 M60 000007A Bio Plex Pro Mouse Cytokine 9 Plex Panel 1 x 96 MDO OOOOO0EL Bio Plex Pro Mouse Cytokine 23 Plex Panel 1 x 96 M60 O009RDPD Bio Plex Pro Mouse Cytokine Th1 Th2 Panel 1 x 96 M60 00003J7 Bio Plex Pro Mouse Th17 Cytokine Panel A 6 Plex 1 x 96 M60 O0007NY Bio Plex Pro Mouse Th17 Cytokine Panel B 8 Plex 1 x 96 171 FA001M Bio Plex Pro Mouse Cytokine Th1 Panel 1 x 96 L60 00004C6 Bio Plex Pro Mouse Cytokine Th2 Panel 1 x 96 L60 OOOUKVT Bio Plex Pro Rat Th1 Th2 Panel 1 x 96 171 K1002M Bio Plex Pro Rat Cytokine 24 Plex Panel 1 x 96 171 K1001M Require reagent kit 171 304070 for vacuum separation or 171 304070M for magnetic separation and a vial of standards 171 X40001 Bio Plex x Plex Assays We Mix Premium custom assay service using the Bio Plex Assay Builder www bio rad com bio plex assaybuilder to select analytes and plate type of inter
7. Prepare and Add Streptavidin PE SA PE 1 While the detection antibodies are incubating use Table 12 or the Calculation Worksheet on pages 37 38 to calculate the volume of SA PE 100x and assay buffer needed Streptavidin PE should be prepared 10 min before use 2 Add the required volume of assay buffer to a 15 ml polypropylene tube 3 Vortex the 100x 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 100x stock adjusted to a final volume of 50 ul in assay buffer Table 12 Preparing 1x SA PE from 100x stock includes 25 excess volume of Wells 100x SA PE ul Assay Buffer pl Total Volume ul 96 60 5 940 6 000 48 30 2 970 3 000 5 After the detection antibody incubation slowly remove and discard the sealing tape 6 Wash the plate three times with 100 ul wash buffer 7 Vortex the diluted 1x SA PE at medium speed for 5 sec Pour into a reagent reservoir and transfer 50 pl 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 30 min at room temperature 23 10 11 12 After the streptavidin PE incubation step slowly remove and discard the sealing tape Wa
8. Prewet the filter plate Skip this step if using a flat bottom plate Prewet the wells using 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 Vortex the diluted 1x coupled beads for 30 sec at medium speed Pour the diluted coupled beads 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 Wash the plate two times with 100 pl Bio Plex wash buffer using the wash method of choice Gently vortex the diluted standards blanks samples and controls if applicable for 5 sec Transfer 50 pl to each well of the assay plate changing the pipet tip after every volume transfer Cover plate with a new sheet of sealing tape and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 2 hr at room temperature RT Note 850 rpm provides equivalent performance to shaker settings recommended in previous manuals 1 100 rom for 30 sec 300 rpm for incubation Prepare and Add Detection Antibodies Instructions are provided for diluting the detection antibodies to a 1x concentration 1 While the samples are incubating use Tables 10 11 or the Calculation Worksheet on pages 37 38 to calculate the volume of detection antibodies and detection antibody diluent needed Detection antibodies should be prepared 10 min before use
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10. up the panel 1 Enter pg ml in the Units field 2 Enter 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 30 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 if the array reader is functioning properly Possible Causes High Inter Assay CV Standards were not reconstituted consistently between assays Reconstituted standards and diluted samples were not stored properly Bottom of filter plate not dry 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
11. 7 Activation of Cell Culture Supernatant and Other Biological Fluids Samples may be run neat after activation neutralization or be diluted as required The appropriate dilution factor should be optimized by the user Ensure a final sample volume after treatment and dilution of at least 125 ul to allow for duplicate wells on the assay plate 1 For example if a 1 4 dilution is desired activate the sample by adding 10 ul acid to 50 ul sample Mix thoroughly and incubate for 10 min at room temperature 2 To neutralize sample add 10 ul base Mix thoroughly Treated sample volume is now 70 ul 3 Dilute to 1 4 final in the same diluent used to prepare the standards In this example starting sample volume was 50 ul and a 1 4 dilution gives 200 ul To reach a final volume of 200 ul add 130 ul diluent to 70 ul treated sample Note Serum containing culture medium may contain high concentrations of TGF B A preliminary measurement of medium alone is recommended to determine baseline levels 6 Prepare Coupled Beads Instructions are provided for diluting the coupled beads to a 1x concentration 1 Use Tables 7 8 or the Calculation Worksheet on pages 37 38 to calculate the volume of coupled beads and assay buffer needed 2 Add the required volume of Bio Plex assay buffer to a 15 ml polypropylene tube 38 Vortex the stock coupled beads at medium speed for 30 sec Carefully open the cap and pipet any liquid trapped in the cap ba
12. Bio Plex Pro TGF 6 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 Prepare Standards Prepare Samples Prepare Coupled Beads N OO FO FR WB LD Run Assay 8 Read Plate Troubleshooting Guide Plate Layout Template Calculation Worksheet Safety Considerations Legal Notices Ordering Information l Oo N O A PN 10 11 12 15 18 20 25 31 36 37 39 39 40 Introduction Transforming Growth Factor TGF f Assays The TGF family of proteins plays an important role in a wide range of cellular functions including proliferation differentiation migration and apoptosis There are three known isoforms of TGF 6 with overlapping functions in normal physiology and in disease states such as cancer TGF B1 promotes TH17 immune cell development and bone growth and remodeling TGF f2 plays a vital role in embryonic development and has been shown to suppress IL 2 dependent T cell tumors TGF 63 regulates cell differentiation adhesion and extracellular matrix formation in embryogenesis and wound healing Bio Plex Pro TGF B assays are magnetic bead based multiplex a
13. ample X or control C in the protocol or results file Check if any interfering components such as heparin based anticoagulant 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 35 Plate Layout Template cL LL OF Oo A Wl lt 36 Calculation Worksheet If using either a premixed panel or one singleplex assay with 20x stocks of beads and detection antibodies 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 X50 ul _ yl b Include 20 excess to ensure enough volume ul x 0 20 i ul c Total volume of 1x coupled beads ul ul i d Volume of 20x coupled beads eae fi 6 ul e Volume of assay buffer required ul 2 ul 2 Determine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x x 25 ul ul b Include 25 excess to ensure enough volume ul x 0 25 a ul c Total volume of 1x detection antibodies ul ul ul d Volume of 20x detection antibodies ae aa ner ose ul e Volume of detection antibody diluent required z i ul ul i ul 3 Determine the volume of 1x streptavidin PE needed a Ea
14. ave 5 Click Enter Controls Info and for user defined controls select an analyte from the pull down menu then enter a description and concentration Repeat for each additional analyte in the assay 6 Click Enter Sample Info and enter sample information and the appropriate dilution factor 27 7 Click Run Protocol and confirm that the assay settings are correct a Refer to Table 13 for the recommended RP1 PMT setting Protocols using alternative PMT settings should be validated by the end user b Confirm 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 DD gates are set to 5 000 Low and 25 000 High In Bio Plex Manager software versions 4 0 4 1 and 4 1 1 check Override Gates and set the DD gate values as indicated c 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 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 Click Run Protocol on the pop up screen select Load Plate and click OK to start acquiring data Use the Wash Between Plates la command after every plate run to reduce the poss
15. ch well requires 50 pl streptavidin PE 1x _ X50 pl _ yl b Include 25 excess to ensure enough volume ins ul x 0 25 ete ul c Total volume of 1x streptavidin PE ul ul ul 12 13 14 d Volume of 100x streptavidin PE required ul 100 ___ y e Volume of assay buffer required ul a ul i ul 14 15 16 37 If mixing singleplex assays with 20x stocks of beads and detection antibodies 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 WI XO 20 _ yl 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 e Volume of 20x coupled beads required from each stock tube ul 20 ul 6 f Total volume of combined bead stocks x ul ul 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 1 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
16. ck into the tube This is important to ensure maximum bead recovery Do not centrifuge the vial doing so will cause the beads to pellet 18 4 Dilute coupled beads to 1x by pipetting the required volume into the 15 ml tube Vortex Each well of the assay plate requires 2 5 ul 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 stock tube If necessary perform the volume transfer in two steps Do not use a 1 000 ul capacity pipet and or a wide bore pipet tip Preparing 1x coupled beads from 20x stock includes 20 excess volume Table 7 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 8 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 19 7 Run Assay Considerations Bring all assay components and samples to room temperature before use a Use calibrated pipets and pipet carefully avoiding bubbles Use new pipet tips for every volume transfer Pay close attention to vortexing shaking and incubation instructions Deviation from the protocol may result in low assay signal and assay var
17. diluent each supplied in the kit The resulting solution is used for reconstitution and subsequent dilution of standards This results in a serum matrix based diluent that mimics the matrix in 1 16 diluted serum and plasma samples Table 4 Summary of recommended diluents for standards Sample Type Diluent for Standard Add BSA Serum and plasma Standard sample diluent mix 1 3 None Culture media with serum Culture media None Culture media serum free Culture media To 0 5 final w v Lavage lysate other fluids Sample diluent To 0 5 final w v At least 0 5 final w v BSA is recommended to stabilize analytes and reduce adsorption to labware 12 Reconstitute a Single Vial of Standards This procedure prepares enough material to run each dilution in duplicate 1 2 Gently tap the vial containing the lyophilized standard Add 500 ul of the appropriate standard diluent see Table 4 Do not use assay buffer to reconstitute the standards Gently vortex the reconstituted standard for 5 sec then incubate on ice for 30 min Be consistent with the incubation time in every assay to ensure best results During the incubation period prepare the samples as instructed in section 5 Prepare Samples Prepare the Standard Dilution Series The following procedure produces an eight point standard curve with a fourfold dilution between each point Pipet carefully using calibrated pipets and use new pipet tips for every v
18. e prime procedure to prime channel 1 with wash buffer 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 11 4 Prepare Standards General Instructions It is essential to prepare standards exactly as described in this section Incorrect preparation may lead to low signal or variable measurements from plate to plate The peel off sticker 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 8 Prepare a Diluent for Standards 1 Refer to Table 4 for recommended diluents based on different sample types As a general rule reconstitute and dilute standards in a diluent similar to the final sample type or sample matrix If samples are serum or plasma mix 1 volume of Bio Plex standard diluent with 3 volumes of Bio Plex sample
19. ed optics to measure the different molecules bound to the surface of the beads In the Bio Plex MAGPIX the entire sample load volume is injected into a chamber where the beads are imaged using LED and CCD technology A high speed digital 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 ae Streptavidin Ox lt i a Sah Bead veo MR 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 A
20. est Assays are supplied as premixed coupled beads and detection antibodies in the all in one kit format 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 Singleplex Sets and Individual Components A host of singleplex sets and individual assay components are available For more information refer to bulletin 5507 or go to www bio rad com bio plex 40 Life Science Group 10024984 Rev A P10024984 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 5044 5699 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 69 65 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 03 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 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026674 55 05 Taiwan 886 2 2578 7189
21. iability Assay incubations are carried out in the dark on a shaker at 850 50 rpm Cover the plate with sealing tape and protect from light with aluminum foil Table 9 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 Bio Plex Pro or Bio Plex Pro Il Handheld Magnet Pro Il Wash Station Wash Station or Vacuum Manifold Assay Step Magnetic Program Vacuum Program Manual Wash Steps Add beads to plate MAG x2 VAC x2 2 x 100 ul Sample incubation MAG x3 VAC x3 3 x 100 ul Detection Ab incubation MAG x3 VAC x3 3 x 100 ul SA PE incubation MAG x3 VAC x3 3 x 100 pl Considerations when Using a Vacuum Manifold 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 Thoroughly blot the bottom of the filter plate with a clean paper towel between each vacuum step to prevent cross contamination 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 over the wells to prevent leaking from the bottom 20 Add Coupled Beads Standards and Samples 1 2 Cover unused wells with sealing tape
22. ibility of clogging the instrument 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 When data acquisition is complete select Shut Down A and follow the instructions 28 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 Remove the buffer with the wash method of choice 3 Add 100 ul assay buffer to each well Cover the filter 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 Data Analysis 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 quan
23. igh Bio Plex 3D Standard enhanced Bio Plex MAGPIX N A use default instrument settings Or similar Luminex based system The Bio Plex Pro TGF f assays were developed on the low PMT setting using the Bio Plex 200 system Protocols using alternative standard dilution series or PMT settings should be validated by the end user 14 5 Prepare Samples General guidelines for preparing different sample types are provided here For more information contact Bio Rad Technical Support 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 1 5 or 2 ml polypropylene microcentrifuge tubes If a multichannel pipet will be used to load the plate then aliquot the required volumes into Titertube micro test tubes Do not freeze diluted samples Important before running the assay samples must be activated and diluted as described on pages 17 18 Serum and 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 For serum allow blood to clot at room temperature for 30 45 min For plasma proceed directly to the centrifugation steps
24. lls for filter plate only Add 50 ul 1x beads to wells Wash 2 x 100 ul Add 50 ul standards blank activated samples incubate 2 hr at RT with shaking at 850 rom Wash 3 x 100 ul Add 25 ul 1x detection antibody incubate 1 hr at RT with shaking at 850 rom Wash 3 x 100 ul Add 50 ul 1x streptavidin PE incubate 30 min at RT with shaking at 850 rom Wash 3 x 100 ul Resuspend in 125 ul assay buffer shake at 850 rpm for 30 sec Read plate on Bio Plex system Important Considerations Instruments and Software The 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 Assay Procedures 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 includes a printed Bio Plex Pro Assay Quick Guide bulletin 10024986 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 Bead regions for all analytes are listed in the Read Plate section Multiplexing Compatibility Do not mix TGF B assays with other Bio Plex assay panels or reagent kits Protocols reagents and sample treatment conditions are not compatible
25. mary of recommended sample diluents and dilution factors Sample Type Diluent Add BSA Sample Dilution Serum and plasma Sample diluent None 1 16 final Culture media with serum Culture media None User optimized Culture media serum free Culture media To 0 5 final User optimized Lavage lysate other fluids Sample diluent To 0 5 final User optimized For example activate 25 ul sample neutralize and bring to a final volume of 400 ul At least 0 5 final w v BSA is recommended to stabilize analytes and reduce adsorption to labware Activation of Serum and Plasma 1 To activate the sample add 1 volume of acid 1 N HCl to 5 volumes of sample For example add 5 ul acid to 25 ul of sample Mix thoroughly and incubate for 10 min at room temperature 2 To neutralize the sample add a volume of base 1 2 N NaQH 0 5 M HEPES equal to the volume of 1 N HCI used In this example add 5 ul base and mix thoroughly Treated sample volume is now 35 ul 3 The recommended dilution is 1 16 of the starting untreated sample volume In this example starting sample volume was 25 ul and a 1 16 dilution gives 400 ul To reach a final volume of 400 ul add 365 yl Bio Plex sample diluent to 35 ul treated sample Note To achieve neutral pH of a sample pH 7 2 to 7 6 the actual volume of base required may vary depending on initial sample pH and the buffering capacity of the sample Verify pH using pH paper before running the assay 1
26. need to optimize the lysis sample dilution and sample activation methods to ensure that assay performance is fit for purpose 1 Prepare the cell or tissue lysates according to the instructions provided with the Bio Plex cell lysis kit catalog 171 304011 or 171 304012 The protease inhibitors factor and factor Il are included in the kit PMSF needs to be added to lysis buffer at a final concentration of 2 mM The lysates should be free of particulate matter Determine the protein concentration of the lysate It may be necessary to test lyse your sample with different volumes of lysing solution to obtain the specified protein concentration range Activate lysates as described under Sample Activation and Dilution Dilute the activated sample in sample diluent 0 5 BSA The appropriate final lysate protein concentration should be determined by the user A starting range to try is 50 to 900 ug ml 16 Note For optimal assay performance it is important to dilute lysates as much as possible to reduce the detergent concentration 4 Store untreated nonactivated lysates at 20 C to 70 C Avoid multiple freeze thaw cycles Sample Activation and Dilution First prepare samples as described above To measure immunoreactive TGF it is necessary to treat all sample types with the following activation procedure Samples should be assayed immediately after the neutralization step Do not activate the TGF f standards Table 6 Sum
27. olume transfer 1 2 3 Label nine 1 5 ml polypropylene tubes S1 through S8 and Blank Add the specified volume of standard diluent to each tube Figure 3 Vortex the reconstituted standards gently for 5 sec before removing any volume Add 128 pl to the S1 tube containing 72 pl of standard diluent Vortex at medium speed for 5 sec then use a new pipet tip to transfer 50 ul from S1 tube to S2 tube Vortex Continue with 1 4 fourfold serial dilutions from tube S2 to S8 as shown in Figure 3 Use reconstituted and diluted standards immediately Do not freeze for future use Continue with 1 4 serial dilutions as shown in Figure 3 Use reconstituted and diluted standards immediately Do not freeze for future use 13 128 50 50 50 50 50 50 50 Transfer Volume pl f En One 72 150 150 150 150 150 150 150 150 Diluent pl Reconstituted Standard S1 s2 s3 S4 s5 s6 s7 s8 Blank Fig 3 Preparing a fourfold dilution series with a single reconstituted standard RP1 PMT Setting for Standard Curves The Bio Plex 200 and 3D systems have two RP1 PMT or photomultiplier tube setting options while the Bio Plex MAGPIX has no PMT and therefore no PMT setting options Instead MAGPIX uses default instrument settings similar to low PMT on the Bio Plex 200 Table 5 Table 5 Overview of PMT setting options on Bio Plex systems Instrument RP1 PMT Bio Plex 100 200 Low h
28. prevent cross well contamination 31 Possible 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 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 2 cleaning needles provided with washer Perform regular rinses to minimize salt buildup 32 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 Filte
29. r plate not shaken enough before incubation steps and prior to reading Reader is clogged Low Signal or Poor Sensitivity Standards reconstituted incorrectly Detection antibody or streptavidin PE diluted incorrectly 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 filter plate at 850 50 rpm for 30 sec before incubation steps and immediately before reading the plate Refer to the troubleshooting guide in the Bio Plex system hardware instruction manual bulletin 10005042 Follow the standard preparation instructions carefully Check your calculations and be careful to add the correct volumes 33 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 Possible Solutions Use standard diluent or diluent similar 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 no
30. r 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 Bio Rad 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 well clear plastic lid and tray Titertube micro test tubes For preparing replicate standards samples and controls prior to loading the plate Bulletin 10024986 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 catalog 300 34377 Bio Rad catalog 170 020100 Bio Rad catalog 171 025001 IKA catalog 320 8000 WIR catalog 57019 600 Bio Rad catalog 732 6470 Bio Rad catalog 166 0610 VistaLab catalog 3054 1002 or VistaLab catalog 3054 1004 VistaLab catalog 3054 1006 Pall Life Sciences catalog 4187 Bio Rad catalog 171 304502 Bio Rad catalog 223 9390 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 6 Assay Workflow Prewet we
31. reach operational temperature Calibrate System 1 Select Calibrate a 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 even if assays will be run at high RP1 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 JE icon 10 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 Pro Il use MAG programs Bio Plex handheld magnetic washer Vacuum filtration Bio Plex Pro II use VAC programs Filter plate Vacuum manifold manual Setting up the Bio Plex Pro or Bio Plex Pro Il Wash Station The wash station does not require calibration however it should be primed before use For more information refer to the Bio Plex Pro and Pro Il wash station quick guide bulletin 5826 1 Install the appropriate plate carrier on the wash station 2 Use th
32. sh the plate three times with 100 ul wash buffer To resuspend beads for plate reading add 125 ul of 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 and slowly remove the sealing tape Ensure that the plate cover has been removed before placing the plate on the reader Remove the sealing tape and read the plate using the settings below Refer to section 8 Read Plate for details Note Reading at alternative PMT settings on the Bio Plex 100 Bio Plex 200 or Bio Plex 3D requires validation by the end user to ensure that results meet the user s acceptance criteria Table 13 Read 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 Default Or similar Luminex based system 24 8 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 v 6 0 and Higher The protocol should be prepared in advance so that the plate is read as soon as
33. ssays designed to measure TGF 61 TGF 62 and TGF B3 in human mouse and rat sample matrices such as serum plasma urine tissue culture supernatant and milk Multiplexing with Bio Plex Pro Assays Bio Plex Pro assays enable researchers to quantify multiple protein biomarkers in a single well of a 96 well plate in just 3 to 4 hours These robust immunoassays require as little as 12 5 ul serum or plasma or 50 ul cell culture supernatant or 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 productivity 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 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 types of molecules in a single well of the 96 well microplate on the Bio Plex 3D system up to 100 different types of molecules on the Bio Plex 200 system and up to 50 different types of molecules on the Bio Plex MAGPIX system On the Bio Plex 200 and Bio Plex 3D systems a dedicated flow cytometer with two lasers and associat
34. t 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 34 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 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 FI 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 FI value if the blank s FI value is greater than either the standard or sample value If this is undesirable then assign wells as a s
35. t the same time a green 532 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 median 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 TGF B1 TGF B2 and TGF B3 assays are available in a convenient kit format that includes assay reagent and diluent components in a single box Table 1 Table 1 Contents of Bio Plex Pro TGF assays 1 x 96 Well Component Format Standard diluent 1 bottle 10 ml Sample diluent 1 bottle 40 ml Assay buffer 1 bottle 50 ml Wash buffer 1 bottle 200 ml Detection antibody diluent 1 bottle 5 ml Streptavidin PE 100x 1 tube Filter and or flat bottom plate 96 well 1 plate Sealing tape 1 pack of 4 Assay Quick Guide 1 booklet Coupled magnetic beads 20x 1 tube Detection antibodies 20x 1 tube Standard 1 vial 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
36. the experiment is complete A protocol file specifies the analytes used in the reading the plate wells to be read sample information the values of standards and controls and instrument settings Bio Plex Manager software contains 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 2 Click Select Analytes and create a new panel Visually confirm the selected analytes and proceed to step 3 a Click the Add Panel button ite in the Select Analytes toolbar Enter a new panel name Select Bio Plex Pro Assay Magnetic from the assay pull down menu If using Bio Plex Manager version 5 0 or lower select MagPlex from the assay pull down menu b Click the Add button Enter the bead region number and name for the first analyte Click Add Continue to repeat for each analyte in the assay 25 For reference bead regions are shown in Table 14 c Click the Add button 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 the Add All button e lf some of the analytes need to be removed from the Selected list highlight
37. them and select Remove If desired it is possible to rename the panel by clicking on Rename Panel and entering a new panel name Table 14 TGF f assay bead regions Analyte Bead Region TGF 1 13 TGF B2 72 TGF B3 66 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 s and drag the cursor over all the wells that contain standards Repeat this process for blanks controls Ce and samples X 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 on the peel off sticker provided with each vial of standards 26 411 123 1 5 PS eaa SO Ok Protocol Settings Plate Formatting 1 Describe Protocol 2 Select Analytes 3 Format Plate 4 Enter Standards Info DO nh ati ts r i a DOC CC COC 5 Enter Controls Info 6 Enter Sample Info Fig 4 Plate formatting b Enter a dilution factor of 4 and click Calculate The concentrations for each standard point will be populated for all analytes in the table c Optional enter the lot number of the vial of standards into the Standard Lot box and click S
38. titation of samples that might otherwise be considered out of range OOR 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 6 0 and earlier versions outliers 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 29 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 3D 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 N Volume 50 ul 3 Refer to Table 13 to select the appropriate PMT setting for your instrument gt A Plate name 96 well plate 5 Analysis type Quantitative 5PL Curve Fit Number of standards 8 Select Analytes to set
39. ul 11 12 f Total volume of combined detection antibody stock ul x ul 12 5 13 g Volume of detection antibody diluent 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 38 Safety Considerations Eye protection and gloves are recommended when using these products Consult the MSDS for additional information The 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 U S government publication Biosafety in Microbiological and Biomedical Laboratories CDC 1999 Legal Notices Acrodisc Acroprep and Supor are trademarks of Pall Corporation MagPlex xMAP xPONENT FLEXMAP 3D 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 39 Ordering Information TGF f Premixed Multiplex Panel Catalog Bio Plex Pro TGF f
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