Mouse Cytokine 20-Plex Panel
Contents
1. cs sisa angan aaa Red ana Seabee Ena ag a A Da ees 28 Explanation of Symbols 4 2 ndn nt m te e nes 29 iii Kit Contents and Storage Storage All components of the Mouse Cytokine 20 Plex Panel are shipped at 2 to 8 C Upon receipt store all kit components at 2 to 8 C Do not freeze Contents The components and amounts included in the Mouse Cytokine 20 Plex Panel are listed below Reagents Provided 100 Test Kit Mouse 20 Plex Antibody Bead Solution 1X contains 7 5 mM 2 5 mL x 1 vial sodium azide Mouse 20 Plex Biotinylated Antibody Concentrate 10X contains 1 mL x 1 vial 0 196 sodium azide Mouse 20 Plex Lyophilized Standard contains 0 196 sodium azide 2 vials Wash Solution Concentrate 20X contains 0 1 sodium azide 15 mL x 1 bottle Assay Diluent contains 0 196 sodium azide 15 mL x 1 bottle Incubation Buffer contains 0 0596 sodium azide 12 mL x 1 bottle Biotin Diluent contains 3 3 mM thymol 12 mL x 1 bottle Streptavidin RPE Concentrate 10X contains 0 1 sodium azide 1 mL x 1 vial Streptavidin RPE Diluent contains 3 3 mM thymol 12 mL x 1 bottle 96 well Filter Plate x 96 well plate iv Overview Purpose Background Information Introduction Invitrogen s Multiplex Bead Immunoassay Kits are developed to maximize flexibility in experimental design permitting the measurement of one or multiple proteins in panels designed by the researcher The Mouse Cytokine 20 Plex Panel contains all the r2. 8 C Protein standards may be analyzed alone or combined with other protein standards for higher levels of multiplexing Do not combine more than 4 vials See page 9 Protocol Summary Pre wet plate Add 25 uL 1X antibody coated beads and 200 uL Wash Solution Wash 1 x 200 uL Sample type Standard Serum plasma tissue culture supernatant Add 50 uL Add 50 uL incubation buffer incubation buffer Add 100 uL Add 50 uL standard assay diluent Add 50 uL sample w x Shake for 2 hr at RT in the dark Wash 2 x 200 uL Add 100 uL 1X detector antibody Shake for 1 hour at RT in the dark Wash 2 x 200 uL Add 100 uL 1X SAV RPE Shake for 30 min at RT in the dark Wash 3x 200 uL Add 100 uL wash buffer Shake for 2 3 min Read in Luminex Detection System Total time 3 5 hr 3 Detector R phycoerythrin Q Protein R A antibody IK RPE 27 Plate Plan Template aeq Jequunw 101 QI 91eld Jequunu Boyeye gt Wy 28 Explanation of Symbols Symbol Description Symbol Description Catalogue Number RUO Research Use Only Batch code In vitro diagnostic medical device Use by aud Manufacturer Temperature limitation 9 lt O m 9 m European Community authorised representative With contains Without does not contain Protect from light Consult accompanying do
3. handling To prepare a 1X Streptavidin RPE stock dilute 10 uL of 10X Streptavidin RPE in 100 of uL Streptavidin RPE Diluent per assay well Each well requires 100 uL of the diluted Streptavidin RPE See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Number of Vol 10X Vol Streptavidin RPE Wells Streptavidin RPE Concentrate Diluent 24 0 24 mL 2 4 mL 32 0 32 mL 3 2 mL 40 0 40 mL 4 0 mL 48 0 48 mL 4 8 mL 56 0 56 mL 5 6 mL 64 0 64 mL 6 4 mL 72 0 72 mL 7 2 mL 80 0 80 mL 8 0 mL 88 0 88 mL 8 8 mL 96 0 96 mL 9 6 mL Continued on next page 15 Assay Procedure Continued Assay Reading 16 10 Remove the liquid from wells by aspiration with the vacuum manifold Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds then aspirate with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of prepared 1X Streptavidin RPE to each well and incubate the plate for 30 minutes at room temperature on an orbital shaker Shaking should be sufficient to keep the beads suspended during incubation 500 600 rpm Remove the liquid from wells by aspiration with the vacuum manifold Wash beads by adding 200 uL Working Wash Solution to the wells allow the beads to soak for 10 seconds then aspirate with the vacuum manifold Repeat this washing step 2 a
4. in the Singleplex Bead Kit s The concentrations of the protein components of the standard are indicated on the Technical Data Sheet Perform standard dilutions in glass or polypropylene tubes When using serum samples reconstitute the standard with Assay Diluent provided If using other sample types e g tissue culture supernatant reconstitute the standard with a mixture composed of 50 Assay Diluent and 50 of the matrix which closely resembles the sample type 50 50 mixture For example When the sample type is RPMI medium containing 5 FBS the standards should be reconstituted in a mixture composed of 50 Assay Diluent and 50 RPMI containing 5 FBS Protein standards may be analyzed alone or combined with other protein standards for higher levels of multiplexing Do not combine more than 4 vials To the standard vial s add the suggested reconstitution volume of the appropriate diluent see table on next page Do not vortex When mixing or reconstituting protein solutions always avoid foaming Replace the vial stopper and allow the vial to stand undisturbed for 10 minutes Gently swirl and invert the vial 2 to 3 times to ensure complete reconstitution and allow the vial to sit at room temperature for an additional 5 minutes Continued on next page Preparing Reagents Continued One vial of standard Reconstitute the standard vial in the suggested reconstitution volume usually 1 mL of appropriate dil
5. is recommended that in house controls be included with every assay If control values fall outside pre established ranges the assay may be suspect Contact Invitrogen Technical Support for product and technical assistance Do not mix or substitute reagents with those from other lots or sources Continued on next page Before Starting Continued Recommended Plate Plan Note e Handle all blood components and biological materials as potentially hazardous Follow standard precautions as established by the Centers for Disease Control and Prevention and by the local Occupational Safety and Health Administration when handling and disposing of infectious agents e This kit contains materials with small quantities of sodium azide Sodium azide reacts with lead and copper plumbing to form explosive metal azides Upon disposal flush drains with a large volume of water to prevent azide accumulation Avoid ingestion and contact with eyes skin and mucous membranes In case of contact rinse affected area with plenty of water Observe all federal state and local regulations for disposal It is recommended that a plate plan be designed before starting the assay A plate plan template is provided on page 28 The following is a suggested plate plan B blank Assay Diluent Standards 7 through 1 lowest concentration to highest The remainder of the plate is available for controls
6. reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 23 References The references below demonstrate the success customers achieve when using Invitrogen Multiplex Assays For a complete list visit www invitrogen com luminex 10 11 Chang D H et al 2005 Sustained expansion of NKT cells and antigen specific T cells after injection of a galactosyl ceramide loaded mature dendritic cells in cancer patients J Exp Med 201 1503 1517 Kinter A et al 2004 CD25 CD4 regulatory T cells from the peripheral blood of asymptomatic HIV infected individuals regulate CD4 and CD8 HIV specific T cell immune responses in vitro and are associated with favorabl
7. whenever possible This calibration assures lot to lot consistency in performance However the concentration of the reconstituted standards may vary with each new lot of standard Therefore it is important to check the concentration of the standard listed on the Technical Data Sheet and to verify all concentration values entered into the data analysis software Improper reconstitution or Check standard reconstitution and dilution of the standard dilution as described on page 9 Continued on next page 20 Troubleshooting Continued Problem Leaky plate Cause Solution remains on the bottom ofthe wells after vacuum aspiration causing wicking and leakage of well contents during next incubation Filter plate membrane tearing Solution After final wash step and plate taps use a clean absorbent towel to blot the bottom ofthe filter plate before addition of next liquid phase or data acquisition step Excessive vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface 21 Appendix Technical Support World Wide Visit the Invitrogen website at www invitrogen com for Web e Technical resources including manuals Technical Data Sheet quick calculation worksheet application notes MSDSs FAQs formulations citations handbooks and more e Complete Technical Suppor
8. 11 Working Wash Solution Required Total Volume of 1X Bead Mixture Volume 10X Beads x of plexes To prepare a 1X Antibody Bead mixture for a multiplexed assay pipette the beads and Working Wash Solution using the volumes calculated with the formulas presented above into a foil wrapped tube Vortex the tube for 30 seconds then sonicate for 30 seconds The mixture is ready to be used in a multiplexed assay If desired premixed beads can be stored overnight at 2 to 8 C Continued on next page 25 Higher Multiplexing Continued Multiplexing Biotinylated Antibodies Multiplexing Standards 26 The volume required for a multiplexed assay can be calculated by using the formulas presented below Total Volume of 1X Biotinylated Antibody Mixture Required 0 110 mL x of wells Volume of each 10X Biotinylated Antibody Concentrate Required Total Volume of 1X Biotinylated Antibody Mixture Required 11 Biotin Diluent Solution Required Total Volume of 1X Biotinylated Antibody Mixture Volume 10X Biotinylated Antibody x of plexes To prepare a Biotinylated Antibody mixture for a multiplexed assay pipette the Biotinylated Antibody Concentrates and Biotin Diluent using the volumes calculated with the formulas presented above into a tube Mix gently The mixture is ready to be used in a multiplexed assay If desired premixed 1X Biotinylated Antibody can be stored overnight at 2 to
9. 2006 Toll like receptor 4 plays a crucial role in the immune adrenal response to systemic inflammatory response syndrome Proc Natl Acad Sci USA 103 16 6392 6397 For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use 24 Higher Multiplexing Introduction Important Multiplexing Antibody Beads Invitrogen s Multiplex Bead Kits are designed to permit maximal flexibility in experimental design These kits can be used alone or combined with other Singleplex Bead Kits to enable the development of higher level multiplexed assays designed by the researcher Instructions for combining bead mixtures and Biotinylated Antibody mixtures in the development of multiplexed assays are presented below Before preparing multiplexed assays it is important to verify that each analyte is represented by a unique bead region This assures the compatibility of each bead in the development of multiplexed assays Up to 10 bead concentrates pre mixed panel multiplexes and or singleplexes can be combined to increase the number of proteins being monitored After loading the 1X 20 Plex bead solution prepare the following The volume required for a multiplexed assay can be calculated by using the formulas presented below Total Volume of 1X Antibody Bead Mixture Required 0 0275 mL x of wells Volume of each 10X Antibody Bead Concentrate Required Total Volume of 1X Antibody Bead Mixture Required
10. 5 minutes rinse the probe and reinstall Run an unclog protocol See instrument manual Readjust the instrument probe height If it is too low it could puncture the well membrane If it is too high air could be pulled up with the liquid which may appear as bead fragments to the instrument Continued on next page 19 Troubleshooting Continued Problem Cause Solution During washing The filter plate is clogged Dislodge the clog by gently pushing steps the vacuum the pointed end of a 15 mL plastic manifold does not conical tube into the bottom of the aspirate the liquid plate under the clogged well This from wells of the procedure clears the small opening filter plate in the plastic casing Dislodge by placing the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well To prevent filter plate clogging clarify samples by centrifugation at 1 000 x g for 10 minutes prior to analysis Some samples may also require filtration prior to analysis Lack of a tight seal Hold the plate firmly against the vacuum manifold to form a tight seal If only a partial plate is being analyzed cover the empty wells with a self adhesive plate seal In house controls Incorrect concentration The standard proteins included in perform differently entered in data analysis Invitrogen s Singleplex Bead Kits in subsequent software are calibrated to NIBSC assays preparations
11. Concentrate 0 24 mL 2 4 mL 0 32 mL 3 2mL 0 40 mL 4 0 mL 0 48 mL 4 8 mL 0 56 mL 5 6 mL 0 64 mL 6 4 mL 0 72 mL 7 2 mL 0 80 mL 8 0 mL 0 88 mL 8 8 mL 0 96 mL 9 6 mL Continued on next page 13 Assay Procedure Continued Analyte Detection 14 After the 2 hour capture bead incubation remove the liquid from wells by aspiration with the vacuum manifold Add 200 uL of Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds then aspirate with the vacuum manifold Repeat this washing step Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of prepared 1X Biotinylated Detector Antibody page 13 to each well and incubate the plate for 1 hour at room temperature on an orbital shaker Shaking should be sufficient to keep the beads suspended during incubation 500 600 rpm Prepare the Luminex 100 or 200 instrument during this incubation step Refer to the Luminex Instrument Quick Reference card provided in kit Refer to the Technical Data Sheet for all bead regions and standard concentration values Ten to fifteen minutes prior to the end of the detector incubation step prepare the Streptavidin RPE and then proceed with Assay Reading Step 1 Continued on next page Assay Procedure Continued Preparing The Streptavidin RPE is supplied as a 10X concentrate and must Streptavidin RPE be diluted prior to use Protect Streptavidin RPE from light during
12. alculation Worksheet for auto calculation of all assay dilutions Continued on next page 11 Assay Procedure Continued Analyte Capture Note Analyte Capture 12 Determine the number of wells required for the assay 2 Use an adhesive plate cover to seal any unused wells This will keep the wells dry for future use 3 Pre wet the designated assay wells by adding 200 uL of Working Wash Solution into designated wells Incubate plate 15 to 30 seconds at room temperature 4 Aspirate the Working Wash Solution from the wells using the vacuum manifold 5 Vortex the 1X Antibody Beads for 30 seconds then sonicate for at least 30 seconds immediately prior to use in the assay 6 Pipette 25 uL of the bead solution into each well Once the beads are added to the plate keep the plate protected from light When multiplexing other proteins with this 20 Plex Panel Aspirate the liquid by gentle vacuum using the vacuum manifold And prepare a 1X Antibody Bead solution refer to Higher Multiplexing instructions on page 25 7 Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds 8 Aspirate the Working Wash Solution from the wells with the vacuum manifold Repeat this washing step 9 Blot the bottom of the filter plate on clean paper towels to remove any residual liquid Note Place the filter plate on a plate cover or non absorbent surface before all incubations Continued on
13. and samples which may be run as a singlet or in duplicate as desired Running all standards samples and controls in duplicate is recommended Preparing Reagents Introduction Preparing Wash Solution Sample Preparation Guidelines Review the information in this section before starting The Mouse Cytokine 20 Plex Panel includes both antibody bead reagents and buffer reagents Prepare components of the Mouse Cytokine 20 Plex Panel according to instructions below Note Bring all reagents and samples to room temperature before use Upon storage at 2 to 8 C a precipitate may form in the 20X Wash Solution Concentrate If this occurs warm the 20X Wash Solution Concentrate to 37 C and mix until the precipitate is dissolved 1 Prepare a IK Working Wash Solution for use with a 96 well plate by transferring the entire contents ofthe Wash Solution Concentrate bottle to a500 mL container or equivalent and then add 285 mL of deionized water Mix well 2 The 1X Working Wash Solution is stable for up to 2 weeks when stored at 2 to 8 C Note To prepare smaller volumes of 1X Working Wash Solution mix part of 20X concentrate with 19 parts of deionized water Mix well e Serum and tissue culture supernatants are suitable for use with Invitrogen s Multiplex Bead Immunoassays Additional sample types may be suitable but have not been thoroughly validated If possible avoid the use of hemolyzed or lipemic sera The appropriate
14. ant signals seen with some sera attributed to heterophilic antibodies Though such samples have not been seen to date the possibility of this occurrence cannot be excluded Troubleshooting Introduction Refer to the table below to troubleshoot problems encountered with the use of Invitrogen s Multiplex Bead Kits on the Luminex platform To troubleshoot problems with the Luminex instrument refer to the manual supplied with the instrument For more troubleshooting solutions visit www invitrogen com luminex Problem Cause During data Bead aggregation analysis insufficient and or erratic bead count is observed Loss of beads due to the filter plate membrane tearing Clog in instrument or probe Probe height set incorrectly Solution Make sure to vortex the beads for 30 seconds and then sonicate the beads for at least 30 seconds prior to beginning the assay to break up any bead aggregates Empty wells and add fresh wash buffer Shake for 2 to 3 minutes to resuspend the beads To prevent membrane tearing place pipette tips on the side of the well rather than straight down onto the membrane when dispensing liquid into the wells Turn the vacuum manifold on before placing the filter plate on the top to prevent vacuum surge When evaluating a new vacuum manifold adjust the vacuum force so that 3 seconds are required to empty 0 2 mL from the wells of a plate Remove probe sonicate for
15. ations washing steps and loading beads are performed in the filter bottom plate supplied with the kit 1 To wash beads place the filter plate on the vacuum manifold and aspirate the liquid with gentle vacuum do not exceed 5 mm Hg Excessive vacuum can cause the membrane to tear resulting in antibody bead loss Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface Stop the vacuum pressure as soon as the wells are empty Do not attempt to pull the plate off the vacuum manifold while the vacuum is still on or filter plate damage may occur Release the vacuum prior to removing the plate If solution remains in the wells during vacuum aspiration do not detach the bottom of the 96 well filter plate In some cases minor clogs in the filter plate may be dislodged by carefully pressing the bottom of the plate under the clogged well with the pointed end of a 15 mL plastic conical tube Place the filter plate on a clean paper towel and use a gloved thumb or a 1 mL Pasteur pipette bulb to plunge the top of the clogged well Empty all clogged wells entirely before continuing the washes Note Do not attempt to repetitively pull vacuum on plates with clogged wells This can compromise the unclogged wells and bead loss may occur After all wells are empty lightly tap or press the filter plate onto clean paper towels hold the plate in the center for tapping to remove exc
16. conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this Assay Product for a full refund prior to using it in any manner You the customer acquire the right under Luminex Corporation s patent rights if any to use this Assay Product or any portion of this Assay Product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen
17. cuments Bux 1 Directs the user to consult instructions for use IFU accompanying the product Copyright Invitrogen Corporation 16 June 2010 29 Notes 6 invitrogen Corporate Headquarters en Corporation n Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country ific contact information visit our web site at www invitrogen com
18. dditional times for a total of 3 washes Blot the bottom of the filter plate on clean paper towels to remove residual liquid Add 100 uL of Working Wash Solution to each well Shake the plate on an orbital shaker 500 600 rpm for 2 to 3 minutes to resuspend the beads Note If the plate cannot be read on the day of the assay cover and store the plate in the dark overnight at 2 to 8 C for reading the following day without significant loss of fluorescent intensity Aspirate Working Wash Solution from stored plates and add 100 uL fresh Working Wash Solution Place the plate on an orbital shaker for 2 to 3 minutes at 500 600 rpm prior to analysis Uncover the plate and insert the plate into the XY platform of the Luminex 100 or 200 instrument and analyze the samples Determine the concentration of samples from the standard curve using curve fitting software It is recommended to use the five parameter algorithm with a weighted function 1 y depending on the software package used Instrument Setup Helpful guides for Luminex 100 and 200 users 1 QVO decus b Ts Assign the appropriate Bead Region refer to the kit specific technical data sheet to each analyte We recommend that the user count 100 events bead region Set Minimum Events to 0 Set Sample Size to 50 ul Set Flow Rate to 60 ul minute For Invitrogen kits we recommend an initial Double Discriminator DD gate setting of 7 800 15 200 This sett
19. e clinical markers of disease status J Exp Med 200 331 343 Pickering A et al 2004 Cytokine response to infection with bacillus anthracis Spores Infect Immun 72 6382 6389 Piqueras B et al 2006 Upon viral exposure myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors Blood 107 6 2613 2618 Raza K et al 2005 Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin Arthritis Res amp Ther 7 4 R784 R795 Rice P et al 2005 Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge J Pharmacol Exp Ther 314 3 1079 1086 Szodoray P et al 2004 Circulating cytokines in primary Sjorens Syndrome determined by a multiplex cytokine system Scand J Immunol 59 592 599 Talwar S et al 2006 Gene expression profiles of peripheral blood leukocytes after endotoxin challenge in humans Physiol Genomics 25 203 215 Wille Reece U et al 2004 Immunization with HIV 1 Gag protein conjugated to a TLR7 8 agonist results in the generation of HIV 1 Gag specific Th1 and CD8 T cell responses J Immunol 172 449 456 Williams D L et al 2005 Modulation of the phosphoinositide 3 kinase pathway alters innate resistance to polymicrobial sepsis J Immunol 174 7676 7683 Zacharowski K et al
20. eagents that are intended for use with the 100 or 200 dual laser detection system manufactured by Luminex Corporation and sold by Invitrogen and other vendors For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use Advances in the field of cell biology have defined a complex and interdependent set of extracellular and intracellular signaling molecules that control normal cell function Therefore there is growing interest among researchers as well as drug discovery groups in simultaneously monitoring multiple components of signaling pathways Solid phase multiplex protein assays are the tools of choice in these studies as they maximize efficiency by simultaneously profiling several proteins within individual samples Invitrogen s Multiplex Bead Immunoassays are solid phase protein immunoassays that use spectrally encoded antibody conjugated beads as the solid support The spectral beads are suitable for use in singleplex assays or may be mixed for multiplex assays according to the researcher s requirements Each assay is carefully designed and tested to assure that sensitivity range and correlation are maximized The assay is performed in a 96 well plate format and analyzed with a Luminex 100 or 200 instrument which monitors the spectral properties of the capture beads while simultaneously measuring the quantity of associated fluorophore Standard curves generated with this assay system extend ov
21. er several orders of magnitude of concentrations while the sensitivity and quantitation of the assays are comparable to ELISAs Enzyme Linked Immuno Sorbent Assays Assay standards are calibrated to NIBSC National Institute for Biological Standards and Controls reference preparations when available to assure accurate and reliable results Continued on next page Overview Background Information Continued Assay Overview 6 lt e e e Antibody Conjugated Beads Fey e lt gt 60x5 exe ex Analyte Capture 15 IFNA x ex Detection Antibody u Kan Analyte Detection Continued Invitrogen s Mouse Cytokine 20 Plex Panel is designed for the quantitative determination of FGF basic GM CSF IFN y IL 1o IL 1 IL 2 IL 4 IL 5 IL 6 IL 10 IL 12 p40 p70 IL 13 IL 17 IP 10 KC MCP 1 MIG MIP 1a TNF a and VEGF in serum and tissue culture supernatant This kit has not been tested for multiplexing with other markers Should user elect to multiplex this kit with other Luminex kits the assay conditions should be determined empirically for each specific application Visit the Invitrogen web site for a current listing of available Invitrogen multiplex bead immunoassays and reagents at www invitrogen com luminex The xMAP technology combines the efficiencies of multiplexing up to 100 different proteins for simultaneous analysis with reproducibility similar to ELISA The technology uses 5 6 um polyst
22. ess fluid from the bottom of the filter plate Do not invert plate Following the last aspiration and plate taps use a clean absorbent towel to blot the bottom of the filter plate before addition of next liquid phase or data acquisition step Do not leave plate on absorbent surface when adding reagents Continued on next page Assay Procedure Continued Reverse To reduce bubbles and loss of reagents due to residual fluid left in Pipetting pipette tips use the recommended reverse pipetting technique Recommendation To reverse pipette set the pipette to the appropriate volume needed Note Do not reverse pipette volumes lt 20 uL 2 Press the push button slowly to the first stop and then press on past it Note the amount past the first stop will depend on the volume of liquid available to aspirate from 3 Immerse the tip into the liquid just below the meniscus Release the push button slowly and smoothly to the top resting position to aspirate the set volume of liquid 5 Place the end of the tip against the inside wall of the recipient vessel at an angle 6 Press the push button slowly and smoothly to the first stop Some liquid will remain in the tip this should not be dispensed 7 Remove the tip keeping the pipette pressed to the first stop Note Bring all reagents and samples to room temperature before use Online Tool Go to http www invitrogen com luminex under Multiplex Solution Tools click Luminex C
23. filter plates are designed to be used in conjunction with a vacuum manifold do not exceed 5 mm Hg and emptied from the bottom Do not freeze any component of this kit Store kit components at 2 to 8 C when not in use Allow all reagents to warm to room temperature before use air warm all reagents at room temperature for at least 30 minutes or alternatively in a room temperature water bath for 20 minutes except plate and standard vials The fluorescent beads are light sensitive Protect the beads from light to avoid photobleaching of the embedded dye Use aluminum foil to cover test tubes used in the assay Cover filter plates containing beads with an opaque or aluminum foil wrapped plate cover Since the amber vial does not provide full protection keep the vial covered in the box or drawer when not in use Do not expose beads to organic solvents Do not place filter plates on absorbent paper towels during loading or incubations as liquid may be lost due to contact wicking An extra plate cover is a recommended surface to rest the filter plate Following plate washing remove excess liquid and blot from the bottom of the plate by pressing the plate on clean paper towels When pipetting reagents maintain a consistent order of addition from well to well to ensure equal incubation times for all wells To prevent filter tearing avoid touching the filter plate membrane with pipette tips Do not use reagents after kit expiration date It
24. ik Mouse Cytokine 20 Plex Panel For simultaneous quantitative determination of FGF basic GM CSF IFN y IL 1a IL 1 IL 2 IL 4 IL 5 IL 6 IL 10 IL 12 p40 p70 IL 13 IL 17 IP 10 KC MCP 1 MIG MIP 1a TNF a and VEGF in mouse serum and tissue culture supernatant Catalog no LMC0006 Rev 1 1 16 June 2010 PRLMCO0006 Table of Contents Tableof Contents achte elle ep Bade ill Kit Contents and Storage iere e Here Wa NG e e ERRARE Mee iv IG trOdUCELIOTI uia oor herus een 1 OVETVIE WA ie er UH RR te Tried eee etti der Weg iore eset isa 1 Methods sasae 4 Before Starting EE ee aA ESRB ERST RSE nae 4 Preparing Reagents 1i eere je tante tree eee ie nee rra nne tna esee de earns 7 ASSAY Procedures P 10 Instrument Setup eee eene Sero ien eoe gu nes gea des e eap ces neve eee eee ena aea des esae eet 17 Performance Characteristics and Limitations of the Procedure 18 Troubleshooting sess eren enter ennt teen eren enne nnne enne 19 jWedbpoem e an 22 Technical Support nennen cuvess sten eene de danni de a eaa dea 22 Purchaser Notification ee detenti rete te e REP telo ep ERR ERE 23 References a Sees reete amer A RU AO IT ERE a aga n Eee re a akah 24 Higher Multiplexing eese eene nnne enne neret nana naen enne 25 Protocol Summaty 2 ro da med E p o eh GAGANA E d e tei idet 27 Plate Plan Template
25. ing may vary among instruments and must be determined by the user Collect Median RFU Note All Invitrogen Multiplex Luminex Kits are qualified at low PMT setting 17 Performance Characteristics and Limitations of the Procedure Performance Characteristics Procedure Limitations 18 Refer to analyte specific Technical Data Sheet for performance claims Do not extrapolate the standard curve beyond the highest or lowest standard point the dose response and data collected in these regions may be non linear and should be considered inaccurate Note In some cases further dilution of the standard beyond 7 points may be possible to extend the low end of the standard curve e Dilute samples that are greater than the highest standard with Assay Diluent or appropriate matrix diluent reanalyze these samples and multiply results by the appropriate dilution factor e Samples are diluted in the assay 1 2 50 uL of sample and 50 uL of Assay Diluent relative to the standards Be sure to account for this dilution factor during sample calculations e The influence of various drugs aberrant sera hemolyzed hyperlipidemic jaundiced etc and the use of biological fluids in place of serum plasma and tissue culture supernatant samples have not been thoroughly investigated The rate of degradation of analytes in various matrices may not have been investigated The immunoassay literature contains frequent references to aberr
26. next page Assay Procedure Continued Analyte Capture Continued Preparing 1X Biotinylated Antibody Number of Wells 24 32 40 48 56 64 72 80 88 96 10 11 12 13 14 Pipette 50 uL Incubation Buffer into each well To wells designated for the standard curve pipette 100 uL of appropriate standard dilution To the wells designated for the sample pipette 50 uL Assay Diluent followed by 50 uL sample to each well or 50 uL in house controls if used Cover filter plate containing beads with an aluminum foil wrapped plate cover Incubate the plate for 2 hours at room temperature on an orbital shaker Shaking should be sufficient to keep beads suspended during the incubation 500 600 rpm Larger radius shakers will need a lower speed and smaller radius shakers will typically handle higher speeds without splashing Ten to fifteen minutes prior to the end of this incubation prepare the biotinylated detector antibody and then proceed to Analyte Detection Step 1 The Biotinylated Antibody is supplied as a 10X concentrate and must be diluted prior to use To prepare a 1X Biotinylated Antibody stock dilute 10 uL of 10X Biotinylated Antibody in 100 uL of Biotin Diluent per assay well Each well requires 100 uL of the diluted Biotinylated Antibody See table below for examples of volumes to combine Note Dilution factor is 1 11 for extra pipetting volume Vol 10X Biotinylated Antibody Vol Biotin Diluent
27. sample types are defined on the Technical Data Sheet included with this multiplex panel e Collect samples in pyrogen endotoxin free tubes Centrifuge separate and transfer samples to polypropylene tubes for storage e Analyze samples shortly after collection or thawing Freeze samples after collection if samples will not be tested immediately Avoid multiple freeze thaw cycles of frozen samples Thaw completely and mix well do not vortex prior to analysis e Clarify all samples by centrifugation 1 000 x g for 10 minutes and or filter prior to analysis to prevent clogging of the filter plates e Inthe event that the sample concentrations exceed the standard curve dilute samples and reanalyze Dilute the serum samples in Assay Diluent and dilute tissue culture supernatants in the corresponding tissue culture medium Continued on next page 7 Preparing Reagents Continued Guidelines for Standard Curve Preparation Important Reconstituting Lyophilized Standards Each kit comes with 2 complete sets of standard vials so that 2 runs on the plate can be made with freshly prepared standards Reconstitute the protein standard within 1 hour of performing the assay All standards are calibrated to NIBSC preparations when available Additional standards are available from Invitrogen custom services Before performing standard mixing and serial dilutions confirm reconstitution volumes on the Technical Data Sheet included
28. t contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 813 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail on techsupport invitrogen com ipinfo invitrogen com eurotech invitrogen com MSDS Requests 22 Invitrogen Corporation 542 Flynn Road Camarillo CA 93012 USA Tel Toll Free 1 800 955 6288 E mail techsupport invitrogen com Material Safety Data Sheets MSDSs are available at www invitrogen com msds Purchaser Notification Limited Use Label License No 330 Luminex Assay Product Limited Warranty By opening the packaging containing this Assay Product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this Assay Product in any manner you are consenting and agreeing to be bound by the following terms and
29. uent Two vials of standards Reconstitute each vial with 0 5 mL of appropriate diluent Combine 300 uL from each vial and mix by gently pipetting up and down 5 to 10 times Three vials of standards Reconstitute each vial with 0 333 mL of appropriate diluent Combine 200 uL from each vial and mix by gently pipetting up and down 5 to 10 times je Ww BS Four vials of standards Reconstitute each vial with 0 250 mL of appropriate diluent Combine 150 uL from each vial and mix by gently pipetting up and down 5 to 10 times Preparing The standard curve is made by serially diluting the reconstituted Standard standard in Assay Diluent for serum samples or a mixture of 50 Curve Assay Diluent and 50 tissue culture medium for tissue culture supernatant samples See below Do not vortex Mix by gently pipetting up and down 5 to 10 times 150 pL 150 uL 150 uL 150 uL 150 uL 150 uL 0S ONE OL Xx US Reconstituted 13 19 127 1 81 1 243 1 729 Standard y y y y y y o o o o o o o Y Ly o oS oS S oS Std1 Std2 Std3 Std4 Std5 Std6 Std7 Serum Assay Diluent Tissue culture 5096 Assay Diluent 5096 Tissue Culture Medium Discard all remaining reconstituted and diluted standards after completing assay Return the Assay Diluent to the kit Assay Procedure Method of Washing 10 Incomplete washing adversely affects assay results Perform all wash steps with the Wash Solution supplied with the kit All phases of the assay including incub
30. xperimental The experimental outline for using the Mouse Cytokine 20 Plex Outline Panel is shown below Add beads Add incubation buffer standard and samples then incubate for 2 hours Add detection antibody then incubate for 1 hour Add streptavidin RPE then incubate for 30 minutes Resuspend and acquire data using Luminex Detection system Methods Before Starting Materials Required but Not Provided Luminex xMAP system with data acquisition and analysis software Invitrogen Cat no MAP0200 contact Invitrogen for instrument and software placement services see page 22 Filtration vacuum manifold for bead washing Pall Cat no 5017 is recommended Sonicating water bath Vortex mixer Orbital shaker small diameter rotation recommended Calibrated adjustable precision pipettes preferably with disposable plastic tips A manifold multi channel pipette is desirable Distilled or deionized water Glass or polypropylene tubes Aluminum foil or opaque 96 well plate cover Invitrogen Cat no PC10 Continued on next page Before Starting Continued Procedural Notes Review the procedural notes below before starting the protocol Before mixing plexes check that each protein is represented by a unique bead region Up to 10 bead concentrates singleplexes or pre mixed multiplexes can be combined to increase the number of proteins measured Do not invert the filter plates during the assay The
31. yrene beads which are internally dyed with red and infrared fluorophores of differing intensities Each bead is given a unique number or bead region allowing differentiation of one bead from another Beads of defined spectral properties are conjugated to protein specific capture antibodies and added along with samples including standards of known protein concentration control samples and test samples into the wells of a filter bottom microplate and where proteins bind to the capture antibodies over the course of a 2 hour incubation After washing the beads protein specific biotinylated detector antibodies are added and incubated with the beads for 1 hour During this incubation the protein specific biotinylated detector antibodies bind to the appropriate immobilized proteins After removal of excess biotinylated detector antibodies streptavidin conjugated to the fluorescent protein R Phycoerythrin Streptavidin RPE is added and allowed to incubate for 30 minutes The Streptavidin RPE binds to the biotinylated detector antibodies associated with the immune complexes on the beads forming a four member solid phase sandwich After washing to remove unbound Streptavidin RPE the beads are analyzed with the Luminex detection system By monitoring the spectral properties of the beads and the amount of associated R Phycoerythrin RPE fluorescence the concentration of one or more proteins can be determined Experimental Overview E
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