- Bio-Rad
Contents
1. Plate Formatting Piate Groupings oh ee ey ey rs re oy te eo ch ra s Standard a OOSA e aog 22 22 30 30 8 G G 7 z lbs 23 23 31 31 OOD le sls z 24 52 32 Blank 2 OOL a J Iz 7 25 25 33 33 E OG 2 2 10 10 18 18 26 26 34 34 X Samples OG a 11 11 19 19 27 27 35 35 6 OLJE 42 12 20 20 28 28 26 26 Q Control H OO 5 5 48 13 21 21 29 29 37 37 re Fig 2 Suggested plate layout For detailed instructions on plate formatting in Bio Plex Manager see Section 8 8 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 prevent2. see o gt Ld Nn o PeREEREE oo nN et ce 4 5 6 Enter Sample Info Fig 6 Plate formatting 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 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 Save 5 Click Enter Controls Info and for user defined controls select an analyte from the dropdown 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 31 7 Click Run Protocol and confirm that the assay settings are correct a The Bio Plex Pro diabetes assays were developed on the high RP1 high PMT setting using the Bio Plex 200 system Protocols using alternative PMT settings should be validated by the end user for example when mixing diabetes assays with cytokine assays 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 siz
3. Bio Plex 100 200 Low high Bio Plex 3D Standard enhanced Bio Plex MAGPIX N A use default instrument settings Or similar Luminex based system The Bio Plex Pro diabetes assays were developed on the high PMT setting using the Bio Plex 200 system Protocols using alternative standard dilution series or low PMT settings should be validated by the end user for example when mixing diabetes assays with cytokine assays Table 7 Table 7 Settings for optimal sensitivity on the Bio Plex 200 system Assay Low RP1 PMT High RP1 PMT Human NHP diabetes Mouse diabetes User validation required Rat diabetes arosa Annn Low RP1 PMT broad High RP1 PMT narrow p 9 range cytokine curve range cytokine curve Human diabetes cytokines Mouse diabetes cytokines User validation required Rat diabetes cytokines e Contact Bio Rad technical support for the most up to date recommendations on PMT settings and cross panel multiplexing compatibility Reconstitute a Single Vial of Diabetes Standards This procedure prepares enough material to run each dilution in duplicate 1 Gently tap the vial containing the lyophilized standard 2 Add 500 ul of the appropriate standard diluent Do not use assay buffer or sample diluent to reconstitute the standards 12 8 Gently vortex the reconstituted standard for 5 sec then incubate on ice for 30 min Be consistent with the incubation time in every as
4. cytokine 10x assay for example human insulin and human IL 6 are listed in Table 17 Preparing 1x detection antibodies from 20x stock includes 25 excess volume Table 15 Premixed panel or one singleplex assay Detection of Wells 20x Detection Antibody Total Antibodies ul Diluent pl Volume pl 96 150 2 850 3 000 Table 16 Mixing two singleplex assays or a premixed panel singleplex assay 20x Detection 20x Detection Detection of Wells Antibodies pl Antibodies pl Antibody Total Singleplex 1 Singleplex 2 Diluent pl Volume pl 96 150 150 2 700 3 000 Table 17 Preparing 1x detection antibodies from two stocks at different concentrations Mixing human insulin 20x with human IL 6 10x is one example 20x Detection 10x Detection Detection of Wells Antibodies pl Antibodies pl Antibody Total Diabetes Cytokines Diluent pl Volume ul 96 150 300 2 550 3 000 Due to differences in dilution factors it is not possible to multiplex adiponectin adipsin human VCAM 1 or ICAM 1 with other diabetes or cytokine assays 5 After incubating the beads samples standards and blank slowly remove and discard the sealing tape 25 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 of the assay plate using a multichannel pipet 8 Cover plate with sealing ta
5. 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 Pe 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 W and wait for the lasers optics to reach operational temperature Calibrate System 1 Select Calibrate Wy 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 X icon 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 4 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 II use MAG programs Bio Plex handheld magnetic washer Vacuum filtration Bio Plex
6. instruction manual bulletin 10005042 Follow the standard preparation instructions carefully Check your calculations and be careful to add the correct volumes 37 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 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 38 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
7. ul ul z 2 Determine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x _ x25ul __ yl b Include 25 excess to ensure enough volume i ul x 0 25 ul c Total volume of 1x detection antibodies i ul ul d Volume of 20x detection antibodies Beit Hye ul e Volume of detection antibody diluent required i ul ul ul 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 ul streptavidin PE 1x x 50 ul ul b Include 25 excess to ensure enough volume a ul x 0 25 oOo ul c Total volume of 1x streptavidin PE ul ul ul 10 11 12 d Volume of 100x streptavidin PE required u 100 ___ yl e Volume of assay buffer required ul yl i ul 12 13 14 42 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 F 1 Determine the volume of 1x coupled beads needed a Each well requires 50 ul coupled beads 1x x 50 ul 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 Enter the number of singleplex sets or analytes tubes that will be multiplexed e Volume of 20x coupled beads required from each stock tube N 20 _ uyl 4 6 f Total volume of combined bead stocks x ul ul 5 6 7 g Volume of assay buffer required ul ul ul 4 7 8 2 D
8. 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 the 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 10 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
9. 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 sample 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 39 Appendix Protease Inhibitors Refer to the recommended protocol below for preparing the inhibitors Materials a DDP IV Inhibitor Sigma Aldrich K4264 Molecular Weight 370 24 Store at 4 C Aprotinin Sigma Aldrich A3428 3 8 TIU mg or 3 900 10 400 KIU mg Store at 4 C Preparation of Protease Inhibito
10. array reader To eliminate this possibility use the validation kit to assist in determining 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 prevent cross well contamination 35 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
11. button If 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 on Rename Panel and entering a new panel name 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 Table 20 Bead regions for available Bio Plex Pro diabetes assays Human NHP Diabetes Mouse Diabetes Rat Diabetes Bead Bead Bead Analyte Region Analyte Region Analyte Region Adiponectin 64 Adiponectin 29 Ghrelin 64 Adipsin 35 Ghrelin 64 GLP 1 62 C Peptide 72 GIP 46 Glucagon 63 Ghrelin 26 GLP 1 62 Leptin 65 GIP 14 Glucagon 63 PAI 1 61 GL P 1 2i Insulin 66 Glucagon 15 Leptin 65 Insulin 12 PAI 1 48 Leptin 78 Resistin 30 PAI 1 61 Resistin 65 Visfatin 22 29 Table 21 Bead regions for compatible Bio Plex Pro cytokine assays Assays shown have bead regions that are non overlapping with diabetes assays Human Cytokines Mouse Cytokines Rat Cytokines Bead Bead Bead Bead Bead Group Region Group Il Region Group Region Group II Region Group Region IL 1B 39 IL 1a 63 IL 1a 53 IL 15 42 IL 1a 21 IL 1ra 25 IL 2Ra 13 IL 1B 19 IL 18 20 IL 1B 28 IL 2 38 IL 3 64 IL 2 36 Basic FGF 25 IL 2 22 IL 4 52 IL 12p40 28 IL 3 18 LIF 45 IL 4 38 IL 5 33 IL 18 42 IL 4 39 M CSF 26 IL 5 52 IL 6 19 HGF 62 IL 5 52 MIG 44 IL 6 56 IL 7 74 IFN a2 20 IL 6 38 MIP 2 27 IL 7 38 IL 8 54 LIF 2
12. 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 build up 36 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 Filter 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
13. provided for diluting the detection antibodies to a 1x concentration When mixing diabetes and cytokine assays keep in mind the stock concentrations of detection antibodies as shown below Table 14 Stock concentration of detection antibodies Stock Concentration of Assay Detection Antibodies Human NHP mouse and rat diabetes 20x Human and mouse cytokines groups II 10x Rat cytokines group 20x Mouse cytokines group III 20X 1 While the samples are incubating use Tables 15 17 to calculate the volume of detection antibodies and detection antibody diluent needed Detection antibodies should be prepared 10 min before use 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 either 1 25 ul 20x stock or 2 5 ul 10x stock adjusted to a final volume of 25 ul in detection antibody diluent 24 For reference Table 15 summarizes volumes required for preparing 1x detection antibodies from a single 20x stock Table 16 summarizes volumes required for preparing 1x detection antibodies from a mix of two 20x stocks volumes required for preparing detection antibodies of one diabetes 20x assay and one
14. um dual filter to prevent instrument clogging 17 For assays other than adiponectin and adipsin dilute plasma fourfold 1 4 by adding 1 volume of sample to 3 volumes of Bio Plex sample diluent for example 40 ul sample 120 ul sample diluent Assay samples immediately or aliquot into single use tubes and store at 70 C Avoid repeated freeze thaw cycles Serum Lis 4 To prepare serum allow blood to clot at room temperature for 30 to 45 min Perform centrifugation at 1 000 x g for 15 min at 4 C and transfer the serum to a clean polypropylene tube To completely remove platelets and precipitates centrifuge again at 10 000 x g for 10 min at 4 C Alternatively carefully filter the samples with a 0 8 0 2 um dual filter to prevent instrument clogging Dilute and handle samples as described in steps 6 and 7 above Tissue Culture Supernatant 1 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 0 5 This is done 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 If high levels of analyte are expected samples can be further diluted in culture media Supplement serum free media with 0 5 BSA final Assay samples i
15. 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 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 800 88 22 88 United Kingdom 020 8328 2000 10 0021 0113 Sig 1212
16. 9 IL 9 33 PDGF BB 35 IL 10 19 IL 9 77 M CSF 67 IL 10 56 VEGF 47 IL 12p40 76 IL 10 56 MIF 35 IL 12p40 76 IL 12p70 78 IL 12p70 75 B NGF 46 IL 12p70 78 Group u Bead IL 13 15 IL 13 Sil TNF B 30 IL 13 37 Region IL 17 T2 IL 15 73 TRAIL 66 IL 17 72 IL 17F 28 IL 18 20 IL 17 76 Eotaxin 74 IL 21 14 EPO 14 Eotaxin 43 G CSF 54 IL 22 15 G CSF 54 Basic FGF 44 GM CSF 73 IL 23p19 61 GM CSF 37 G CSF 57 IFN y 34 IL 25 67 GRO KC 57 GM CSF 34 KC 57 IL 27p28 43 IFN y 34 IFN y 21 MCP car 51 IL 31 29 M CSF 26 IP 10 48 MIP 1a 77 IL 33 13 MIP 1a T MCP 1 53 MIP 1 15 MIP 3a 12 MIP 2 27 MIP 10 655 RANTES 55 MIP 30 36 MIP 1B 18 TNF a 21 RANTES 55 PDGF BB 47 TNF a 43 RANTES 37 VEGF 47 TNF a 36 MCP 1 51 VEGF 45 Mouse group Ill cytokines have not been tested for cross reactivity with diabetes or other panels 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 6 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 lt gt controls c and samples X 30 biS 2 vl k Olle n E 4 er N 1 11 12 A 30 30 BEEE REE 25 25 33 33 BARE BARR BREE EI S a Oo OO O n E Es N Jae
17. Bio Plex Pro Diabetes 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 2 Prepare Instrument Prepare Wash Method io Prepare Standards 4 5 Prepare Samples 6 Prepare Coupled Beads 7 Run Assay 8 Read Plate Troubleshooting Guide Appendix Protease Inhibitors Plate Layout Template Calculation Worksheet Safety Considerations Legal Notices Ordering Information i N O O A N 10 11 16 20 22 28 35 40 41 42 46 46 47 Introduction Bio Plex Pro diabetes assays are magnetic bead based multiplex assays designed to measure multiple diabetes related biomarkers in a minimal volume of matrix such as serum plasma tissue culture supernatant or other biological fluids The biomarkers chosen for these assays are circulating proteins involved in the regulation of glucose metabolism These multiplex assays are configured for the detection of various metabolic markers in human mouse and rat samples The multiplex assays for human matrices were tested and found to be cross reactive to at least four common non human primate NHP species including rhesus cynomolgus baboon and marmoset The deg
18. Diabetes Standard Standard 72 ul 150 150 150 150 150 150 150 150 Diluent pl Standard Diluent S1 Tube 200 pl Total 2 s3 S4 S5 S6 S7 S8 Blank 15 5 Prepare Samples General guidelines on preparing samples derived from serum plasma and tissue culture supernatant are provided here including the use of protease inhibitors with plasma samples Once thawed keep samples on ice Prepare dilutions just prior to the start of the assay and equilibrate to room temperature before use a 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 a Do not freeze diluted samples Table 8 Summary of recommended sample diluents and dilution factors Sample Type Diluent Add BSA Sample Dilution Serum and plasma Sample diluent None Fourfold 1 4 Culture media with serum Culture media None User optimized Culture media serum free Culture media To 0 5 final User optimized For Adiponectin Assay Serum and plasma Serum based None Human 1 400 diluent Mouse 1 1600 NHP 1 1600 For Adipsin Assay Serum and plasma Serum based None Human 1 400 diluent Note certain sample types may require a different dilution factor 16 Protease Inhibitors In general diabetes biomarkers are detectable in EDTA treated plasma Free
19. Plex Manager software as instructed in section 8 For users who wish to mix assays from different panels such as diabetes assays with group cytokines guidance is provided here for mixing 2 different lyophilized standards Bead regions were chosen to avoid overlap whenever possible However performance of multiplexes containing assays from different groups have not been extensively validated Therefore users must confirm that the assay performance is still fit for their purpose Selecting a Diluent for Standards Refer to Table 5 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 Table 5 Summary of recommended diluents for standards Sample Type Diluent for Standards Add BSA Serum and plasma Standard diluent None Culture media with serum Culture media None Culture media serum free Culture media To 0 5 final For Adiponectin and or Adipsin Assays Serum and plasma Serum based diluent None 11 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 6 Table 6 Overview of PMT setting options on Bio Plex systems Instrument RP1 PMT
20. T narrow range cytokine standard curve add 12 8 pl of the reconstituted cytokine standard and 128 ul of the reconstituted diabetes standard to the S1 tube for a total volume of 200 ul Figure 4 Vortex at medium speed for 5 sec 4 Add 150 ul of standard diluent to the remaining tubes as shown in Figure 4 Fig 4 S1 mixture and fourfold dilution series of diabetes and cytokine standards for detection at high PMT Produces a narrow range cytokine standard curve Transfer 50 50 50 50 50 50 50 Volume pl 128p 128 pl DVN MSY a a Yi Ya Y e J 500 pl Reconstituted Diabetes Standard 59 2 ul 150 150 150 150 150 150 150 150 500ul C Reconstituted Standard Diluent 1 Tube 200 pl Total s2 s3 s4 s5 s6 s7 s8 Blank Cytokine Standard Diluent pl 5 For low PMT broad range cytokine standard curve add 64 ul of each standard to the S1 tube total volume 200 ul Figure 5 Vortex for 5 sec 6 Use anew pipet tip to transfer 50 ul from the S1 tube to the S2 tube containing standard diluent 7 Continue with 1 4 fourfold serial dilutions from tube S2 to S8 as shown in Figure 5 Fig 5 S1 mixture and fourfold dilution series of diabetes and cytokine standards for detection at low PMT Produces a broad range cytokine standard curve Transfer 50 50 50 50 50 50 50 Volume pl 64 ul 64 ul aa WV NE MEY 250ul 250 ul Reconstituted Reconstituted Cytokine
21. ay system includes fluorescently labeled microspheres and instrumentation licensed to Bio Rad Laboratories Inc by the Luminex Corporation 46 Ordering Information Premixed All in One Multiplex Assays Description Catalog Bio Plex Pro Human Diabetes 10 plex Assay 1 x 96 well 171 A7001M Bio Plex Pro Mouse Diabetes 8 plex Assay 1 x 96 well 171 F7001M Bio Plex Pro NHP Diabetes 11 plex Assay 1 x 96 well 171 W7001M 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 ready for you to mix 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 47 Life Science Group 10010747 Rev D P10010747 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 31 884 0 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
22. d cytokine assays Note that rat diabetes and cytokine standards are premixed into one standard vial Therefore no extra mixing is required Two mixing scenarios are provided in Figures 4 and 5 for detection at high and low PMT respectively One results in a narrow range cytokine standard curve for detection at high RP1 PMT the other gives a broad range cytokine standard curve for detection at low RP1 PMT setting 1 Gently tap both vials of lyophilized diabetes and cytokine standards 2 For high PMT setting narrow range cytokine standard curve add 500 ul of the appropriate standard diluent to each vial For low PMT setting broad range cytokine standard curve add 250 ul of diluent to each vial Do not use assay buffer or sample diluent to reconstitute the standards 8 Gently vortex the reconstituted standards for 5 sec then incubate on ice for 30 min Be consistent with the incubation time in every assay to ensure best results 4 During the incubation period prepare the samples as instructed in the Prepare Samples step Preparing Serial Dilutions Pipet carefully with calibrated pipets and use new pipet tips for every volume transfer 1 Label nine 1 5 ml polypropylene tubes S1 through S8 and Blank 2 For high PMT narrow range cytokine standard curve add 59 2 ul of standard diluent to the S1 tube For low PMT broad range cytokine standard curve add 72 ul of standard diluent to S1 Figures 4 and 5 14 8 For high PM
23. e 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 command after every plate run to reduce the possibility 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 and follow the instructions 32 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 wi
24. eet of sealing tape Avoid pressing down over the wells to prevent leaking from the bottom 22 Add Coupled Beads Standards Blanks Samples and Controls 1 Cover unused wells with sealing tape 2 Prewet the filter plate Skip this step if using a flat bottom plate a Prewet the wells with 100 ul of 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 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 4 Wash the plate two times with 100 pl Bio Plex wash buffer using the wash method of choice 5 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 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 room temperature RT Note 850 rpm provides equivalent performance to previously recommended shaker settings 1 100 rom for 30 sec 300 rpm for incubation Note Be consistent with this incubation time for optimal assay performance and reproducibility 23 Prepare and Add Detection Antibodies Instructions are
25. es 20x Human and mouse cytokine groups I II analytes 10x 10 9 8 7 6 5 Enter the number of wells to be used in the assay 1 Enter the number of diabetes tubes either singleplex or multiplex that will be mixed Enter the number of cytokine tubes either singleplex or multiplex that will be mixed 3 1 Determine the volume of 1x diabetes and cytokine coupled beads needed a Each well requires 50 ul of coupled beads 1x x 50 ul ul b Include 20 excess to ensure enough volume ul x 0 20 ul c Total volume of 1x coupled beads r ul ul ul d Volume of 20x beads required from each diabetes tube s ul 20 ul e Volume of 10x beads required from each cytokines tube s ul 10 ul f Total volume of diabetes bead stock required ul x 5 z ul g Total volume of cytokine bead stock required z ul x A ul h Total volume of combined bead stocks required z ul ul i Volume of assay buffer required ul ul 44 2 Determine the volume of 1x diabetes and cytokine detection antibodies needed a Each well requires 25 ul of detection antibodies 1x x25 pl ul 1 13 b Include 25 excess to ensure enough volume ul x 0 25 ul 13 14 c Total volume of 1x detection antibodies ul ul ul 13 14 15 d Volume of 20x detection antibodies required from each diabetes tube s ul 20 ul 15 16 e Volume of 10x detec
26. es Use new pipet tips for every volume transfer a Pay close attention to vortexing shaking and incubation instructions Deviation from the protocol may result in low assay signal and assay variability 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 13 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 or Pro Il Wash Station Wash Station 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 3x 100 ul Detection Ab incubation MAG x3 VAC x3 3 x 100 ul SA PE incubation MAG x3 VAC x3 3 x 100 ul 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 sh
27. etermine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x x 25 l ul 1 9 b Include 25 excess to ensure enough volume ulxO0 25 sul 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 Hl 20s l 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 43 If mixing diabetes assays 20x bead and detection antibody stocks with cytokine assays 10x stocks follow these directions Note Refer to Table 20 for the maximum number of diabetes and cytokine assays that may be multiplexed Mixing across panels is not applicable to NHP diabetes assays Table 20 Maximum number of singleplex diabetes and cytokine analytes that may be multiplexed Human mouse and rat diabetes 0 2 A 6 8 10 Mouse cytokine group Ill analyt
28. idelines 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 Volume 50 ul Refer to Table 19 to select the appropriate PMT setting for your instrument Plate name 96 well plate Analysis type Quantitative 5PL Curve Fit Number of standards 8 Select Analytes to set 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 34 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
29. ination 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 a On the Bio Plex 200 and Bio Plex 3D systems a dedicated flow cytometer with two lasers and associated 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 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 es Streptavidin Ox lt i zk sauce Bead ven oe Fluore
30. mmediately or aliquot and store at 70 C 18 Lavage Sputum and Other Biological Fluid Samples Keep all samples on ice until ready for use 1 If dilution is required use Bio Plex sample diluent with 0 5 BSA final 2 Centrifugation at 10 000 x g for 10 min at 4 C may be needed to clarify the sample Sample Dilution for Adiponectin and Adipsin Assays Note Physiological levels of adiponectin and adipsin are typically found at high concentrations therefore higher sample dilutions are required to achieve measurable concentrations within the standard curve 1 For human adiponectin and adipsin assays dilute serum or plasma 1 400 in serum based diluent with two 1 20 serial dilutions First dilution 10 ul sample 190 ul serum based diluent a Second dilution 10 ul from the first dilution 190 ul serum based diluent 2 For mouse and NHP adiponectin assays dilute serum or plasma 1 1 600 in serum based diluent with two 1 40 serial dilutions m First dilution 10 ul sample 390 ul serum based diluent a Second dilution 10 ul from the first dilution 390 ul serum based diluent 19 6 Prepare Coupled Beads Instructions are provided for diluting the coupled beads to a 1x concentration When mixing diabetes and cytokine assays keep in mind the stock concentrations of coupled beads as listed in Table 9 Table 9 Stock concentration of coupled beads Assay Stock Concentration of Coupled Beads Human NHP mouse a
31. n adiponectin adipsin assays 40 ml 80 ml Serum based diluent adiponectin adipsin assays only 70 ml N A Assay buffer 50 ml 500 ml Wash buffer 200 ml 15 Detection antibody diluent 5 ml 50 ml Streptavidin PE 100x 1 tube 1 tube Filter and or flat bottom plate 96 well 1 plate 10 plates Sealing tape 1 pack of 4 10 packs of 4 Assay Quick Guide 1 booklet Coupled magnetic beads 20x 1 tube 1 tube Detection antibodies 20x 1 tube 1 tube Standard 1 vial 10 vials Volumes shown are approximate Quantities in Bio Plex Express assays will vary 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 materials Item Ordering Information Bio Plex Pro Assays Quick Guide 2 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 bo
32. ncubation step slowly remove and discard the sealing tape 10 Wash the plate three times with 100 pl wash buffer 11 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 the plate 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 12 Remove the sealing tape and read the plate using the settings below Note Reading at alternative PMT settings on the Bio Plex 100 200 or Bio Plex 3D for example when mixing diabetes assays with cytokine assays requires validation by the end user to ensure that results meet the user s acceptance criteria Table 19 Read the plate using the appropriate instrument settings Instrument RP1 PMT DD Gates Bead Events Bio Plex 100 200 High 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 27 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 a
33. nd 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 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 version 6 0 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 ise 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 For reference bead regions are shown in Table 20 28 Click the Add button when the last analyte has been added and click OK to save the new panel 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
34. nd rat diabetes 20x Human and mouse cytokines groups II 10x Rat cytokines group 20x Mouse cytokines group III 20X Note When using 10 pack reagents ensure that only the required volumes of coupled beads detection antibodies streptavidin PE and buffers have been removed from the tubes or bottles For example transfer a one time volume of assay buffer sufficient to perform all steps of the assay procedure that is prewetting the filter plate diluting coupled beads diluting streptavidin PE and resuspending the beads into a 50 ml reservoir 1 Use Tables 10 12 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 3 Vortex the stock 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 plate requires either 2 5 ul 20x stock or 5 0 ul 10x stock adjusted to a final volume of 50 ul in assay buffer 20 5 Protect the beads from light with aluminum foil Equilibrate to room temperature prior to use Table 10 summarizes volumes required for preparing 1x beads from a single 20x stock Table 11 summarizes volumes required fo
35. ottom microplate for calibrating vacuum manifold 5 Assay Workflow Prewet wells for filter plate only Add 50 ul 1x beads to wells Wash 2 x 100 pl Add 50 ul standards blank samples incubate 1 hr at RT with shaking at 850 rpm Wash 3 x 100 ul Add 25 ul 1x detection antibody incubate 30 min at RT with shaking at 850 rom Wash 3 x 100 ul Add 50 ul 1x streptavidin PE incubate 10 min at RT with shaking at 850 rpm Wash 3 x 100 pl 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 diabetes 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 10024973 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 For human and mouse the maximum number of singleplex diabetes and cy
36. pe and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 30 min at room temperature Prepare and Add Streptavidin PE SA PE 1 While the detection antibodies are incubating use Table 18 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 38 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 vial 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 18 shows an example calculation Table 18 Preparing 1x SA PE from 100x stock includes 25 excess volume of Wells 100x SA PE pl Assay Buffer pl Total Volume ul 96 60 5 940 6 000 26 5 After the detection antibody incubation slowly remove and discard the sealing tape 6 Wash the plate three times with 100 pl 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 sealing tape and protect from light with aluminum foil Incubate on shaker at 850 50 rpm for 10 min at room temperature 9 After the streptavidin PE i
37. r preparing 1x beads from a mix of two 20x stocks volumes required for preparing beads of one diabetes 20x assay and one cytokine 10x assay for example human insulin and human IL 6 are listed in Table 12 Note To minimize volume loss use a 200 300 il 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 wide bore pipet tip Preparing 1x coupled beads from 20x stock includes 20 excess volume Table 10 Premixed panel or one singleplex assay 20x Assay Total of Wells Beads ul Buffer pl Volume ul 96 288 5 472 5 760 Table 11 Mixing two singleplex assays or a premixed panel singleplex assay of Wells 20x beads pl 20x beads ul Assay Total Singleplex 1 Singleplex 2 Buffer pl Volume pl 96 288 288 5 184 5 760 Table 12 Preparing 1x beads from two stocks at different concentrations Mixing human insulin 20x with human IL 6 10x is one example 20x 10x of Wells Beads ul Beads ul Assay Total Diabetes Cytokines Buffer pl Volume pl 96 288 576 4 896 5 760 Due to differences in dilution factors it is not possible to multiplex adiponectin adipsin human VCAM 1 or ICAM 1 with other diabetes or cytokine assays 21 7 Run Assay Considerations a Bring all assay components and samples to room temperature before use Use calibrated pipets and pipet carefully avoiding bubbl
38. ree of cross reactivity was profiled according to the ability of each assay to detect these metabolic markers in the sera and mitogen stimulated peripheral blood mononuclear cell PBMC culture supernatant of these animals The high level of gene homology across the major NHP species may allow the NHP diabetes assays to be used in other species such as chimpanzees African green monkey and pigtail macaques However assay performance has not been specifically evaluated in these animals Cross Reactivity with Non Human Primates African green Pigtail Rhesus Cynomolgus Baboon Marmoset Chimpanzee monkey macaque Tested Not tested Bio Plex Pro assays enable researchers to quantify multiple protein biomarkers in a single well of a 96 well plate in 3 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 discrim
39. rs Stock Solution di Preparation of 10 mM DDP IV inhibitor solution a Weigh 18 5 mg of DDP IV inhibitor lox Dissolve completely in 5 ml of 0 9 NaCl c Aliquot and store at 20 C Avoid repeated freeze thaw cycles Preparation of 1 3 Aprotinin a Weigh 100 mg Aprotinin b Dissolve completely in 7 5 ml of 0 9 NaCl c Aliquot and store at 20 C Avoid repeated freeze thaw cycles Blood Sample Preparation ule 2 Collect whole blood in plasma collection tubes Add 10 ul of 10 mM DDP IV inhibitor per 1 ml of whole blood for a final concentration of 100 uM Add 10 ul of 1 3 Aprotinin per 1 ml of whole blood for a final amount of gt 500 KIU per 1 ml of whole blood Invert the tubes several times to mix the protease inhibitors with blood 40 Plate Layout Template cL LL OF m Oo A WU A lt 41 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 x 50 ul ul b Include 20 excess to ensure enough volume ul x 0 20 Aa ul c Total volume of 1x coupled beads ul ul ul d Volume of 20x coupled beads cae ee _ ul e Volume of assay buffer required
40. say to ensure best results 4 During the incubation period prepare the samples as instructed in the Prepare Samples section Prepare Diabetes Standard Dilution Series from a Single Antigen Vial 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 volume transfer 1 Label nine 1 5 ml polypropylene tubes S1 through S8 and Blank 2 Add the specified volume of standard diluent to each tube Figures 3 and 4 8 Vortex the reconstituted standards gently for 5 sec before removing any volume Add 128 ul into the S1 tube containing 72 ul 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 4 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 Fig 3 Preparing a fourfold dilution series of diabetes standards 128 50 50 50 50 50 50 50 Transfer Volume pl WAPAPRAAAE lt E e _ Reconstituted 72 150 150 150 150 150 150 150 150 Diluent ul Diabetes Standard s1 s2 s3 S4 s5 s6 s7 s8 Blank 13 Please skip this section if not mixing diabetes with cytokine assays Reconstituting Standards for Cross Panel Plexing Follow these directions when mixing human or mouse diabetes an
41. scent 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 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 Bio Plex Pro diabetes assays are offered 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 diabetes assays 1 x 96 Well 10 x 96 Well Component Format Format Standard diluent not included in adiponectin adipsin assays 10 ml 100 ml Sample diluent not included i
42. th 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 quantitation 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 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 33 Luminex xPONENT Software Although gu
43. tion antibodies required from each cytokines tube s ul 10 ul 15 17 f Total volume of diabetes detection antibodies stock required ul x ul 16 2 18 g Total volume of cytokine detection antibodies stock required ul x ul 17 3 19 h Total volume of combined detection antibodies required ul ul 18 19 20 i Volume of detection antibody diluent required ul ul ul 15 20 21 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 ul of streptavidin PE 1x x 50 pl 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 45 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 and Luminex are trademarks of Luminex Corporation The Bio Plex suspension arr
44. tokine analytes that may be mixed is limited by the 10x cytokine antibody stock concentrations as shown in the table below Table 3 Maximum number of singleplex diabetes and cytokine analytes that may be multiplexed Human mouse and rat diabetes Mouse cytokine group III analytes 20x Human and mouse cytokine groups II 10 9 8 analytes 10x 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 41 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 9 11 14 16 and 17 respectively or the Calculation Worksheet on page 43 2 Legend Si 2 ws e Ok Protocol Settings
45. ttom plates 40 x 96 well For magnetic separation on the Bio Plex Pro wash station 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 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 10024973 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 20100 Bio Rad catalog 171 025001 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 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 b
46. zing plasma immediately after preparation and keeping samples frozen until use should provide adequate protection from degradation However users may choose to add protease inhibitors as a precautionary measure Note Protease inhibitors are recommended for use with plasma samples only not with serum Either protease inhibitors may be added to samples at the time of blood collection see protocol in the Appendix or blood samples may be collected directly into a BD P800 collection tube Becton Dickinson catalog 366420 or 3866421 Note BD P800 tubes are not designed for use with mouse samples therefore protease inhibitors if required should be added at the point of blood collection Plasma K EDTA treated plasma is acceptable as long as the sample is immediately frozen upon collection Avoid using heparin treated plasma as it may absorb certain soluble proteins Avoid using hemolyzed samples as this may lead to false positive results 1 Draw whole blood into collection tubes containing anticoagulant 2 If desired add protease inhibitors See protocol in the Appendix 8 Invert tubes several times to mix with either the anticoagulant or the protease inhibitors 4 Perform centrifugation at 1 000 x g for 15 min at 4 C and transfer the plasma to a clean polypropylene tube 5 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
Download Pdf Manuals
Related Search