Cytokine Monkey Magnetic 28-Plex Panel
Contents
1. liquid from the wells by setting the plate on the magnetic separator for 90 seconds then aspirate the well contents with the automated plate washing equipment Continued on next page Assay Procedure Continued Reverse Pipetting Recommendation Note To reduce bubbles and loss of reagents due to residual fluid left in pipette tips use the recommended reverse pipetting technique l To reverse pipette set the pipette to the appropriate volume needed Note Do not reverse pipette volumes 20 uL 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 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 Drag the tip up the side of the tube or reservoir to remove excess volume from the outside of the tips Place the end of the tip against the inside wall of the recipient vessel at an angle above the fluid level Press the push button slowly and smoothly to the first stop Some liquid will remain in the tip this should not be dispensed Remove the tip keeping the pipette pressed to the first stop and return to step 3 above if reusing tips and contamination is not an issue Bring all reagents and samples to room temperature before use Continued on next page 13 Assay Procedure Conti2. 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 1X Working Wash Solution for use with a 96 well plate by transferring the entire contents of the Wash Solution Concentrate bottle to a 500 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 1 part of 20X concentrate with 19 parts of deionized water Mix well e Serum plasma 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 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
3. 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 Experimental The experimental outline for using the Cytokine Monkey Outline Magnetic 28 Plex Panel is shown below NOTE Prewet step required only with the filter bottom plate Prewet wells 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 Luminex 200 Invitrogen Cat no MAP0200 or FlexMAP 3D Invitrogen Cat no FM3D000 contact Invitrogen for instrument and software placement services see page 24 Washing equipment This kit may be used with a filtration vacuum manifold for bead washing Invitrogen EveryPrep Cat no K2111 01 is recommended Alternatively a magnetic separator may be used Dynal MPC 96S Invitrogen Cat no 120 27 This kit is also compatible with automated magnetic bead washing equipment Sonicating water bath Vortex mixer Orbital shaker small diameter rotation recommended Calibrated ad
4. 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 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 17 Assay Procedure Continued Assay Reading 18 Remove the liquid from wells with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Wash the plate by adding 200 uL of Working Wash Solution to the wells Allow the beads to soak for 30 seconds Remove the liquid with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Repeat this washing step for a total of 2 washes The bottom of the filter plate should be blotted on clean paper towels to remove residual liquid after the second wash Add 100 uL of prepared 1X Streptavidin RPE to each well an
5. Proc Natl Acad Sci USA 103 16 6392 6397 For Research Use Only CAUTION Not for human or animal therapeutic or diagnostic use 26 Protocol Summary NOTE Prewet step required only with the filter bottom plate Pre wet plate Add 25 uL 1X antibody coated beads and 200 uL Wash Solution Wash 1 x 200 uL 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 a x Shake for 2 hr at RT in the dark Wash 2 x 200 pL Add 100 uL 1X Biotinylated Detector Antibody Shake for 1 hr at RT in the dark Wash 2 x 200 uL Add 100 pL 1X SAV RPE Shake for 30 min at RT in the dark wash 3 x 200 uL Add 125 uL wash buffer Shake for 2 3 min Read in Luminex xMAP system Total time 3 5 hr Q Protein R m 27 Plate Plan Template a1eq Jequinw 107 QI eld Jequunu ojee Uy 28 Explanation of Symbols Symbol Description Symbol Description REF Catalogue Number Batch code Research Use Only In vitro diagnostic medical device Use by Manufacturer EC REP European Community authorised representative Temperature limitation i ual Without does not contain With contains voten from i Protect from light A Consult accompanyin
6. bent 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 11 Assay Procedure Continued Washing Methods Continued 12 Magnetic Separator Method 1 Set the plate on a magnetic separator for 90 seconds to allow immobilization of the magnetic beads Next aspirate the liquid from the wells using a multichannel pipette Refill the wells with washing solution remove the plate from the magnet then allow the well contents to soak for 60 seconds Again prepare to remove the liquid from the wells by setting the plate on the magnetic separator for 90 seconds then aspirate the liquid contents of the wells with a multichannel pipette Guidelines for Automated Plate Washers l Some optimization of the automated plate washer set up may be required As with manual washing with a magnetic separator the program used for automated washing should include a 90 second period in which the beads are immobilized onto a magnetic separator After the beads are immobilized liquid may be aspirated using automated washing equipment A suggested probe height of 4 8 mm is recommended As with the manual washing method the wells should then be refilled with washing solution the plate removed from the magnet and the contents of the wells allowed to soak for 60 seconds Again prepare to remove the
7. bes used in the assay Cover microplates 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 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 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 Hea
8. bsorbent surface before all incubations Continued on 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 8 Pipette 50 uL Incubation Buffer into each well To wells designated for the standard curve pipette 100 uL of appropriate standard dilution 10 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 11 Cover microplate 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 12 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 Bio
9. ction 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 2006 Toll like receptor 4 plays a crucial role in the immune adrenal response to systemic inflammatory response syndrome
10. curve fitting software It is recommended to use the five parameter logistic algorithm with a weighted function 1 y depending on the software package used Instrument Setup Helpful guides for Luminex 100 and 200 users with xPONENT software l 2 Ww 5 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 Sample Size to 75 uL For Invitrogen kits using MagPlex beads we recommend that the doublet discriminator gates be set at 7 800 20 000 as the initial setting Adjustment of these values may be required for individual instruments Collect Median Fluorescent Intensity MFI Note Use the default setting low PMT for the Luminex 100 and 200 Helpful guides for Luminex FlexMAP 3D users l Ww 2 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 Sample Size to 75 uL For Invitrogen kits using MagPlex beads we recommend that the doublet discriminator gates be set at 7 800 20 000 as the initial setting Adjustment of these values may be required for individual instruments Collect Median Fluorescent Intensity MFI Note Use the default setting high PMT for the Flex MAP 3D 19 Performance Characteristics and Limitations of the Procedure Performance Characteristic
11. d 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 with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Wash the plate by adding 200 uL of Working Wash Solution to the wells Allow the beads to soak for 30 seconds Remove the liquid with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Repeat this washing step 2 more times for a total of 3 washes The bottom of the filter plate should be blotted on clean paper towels to remove residual liquid after the second wash Add 125 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 125 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 100 200 or FlexMAP 3D instrument and analyze the samples Determine the concentration of samples from the standard curve using
12. e spectral properties of the capture beads while simultaneously measuring the quantity of associated fluorophore Standard curves generated with this assay system extend over 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 ec 0 e e Antibody Conjugated Beads 0x5 e 0x3 x Analyte Capture x gt I2 E e 0x Detection Antibody eor un Analyte Detection Continued Invitrogen s Cytokine Monkey Magnetic 28 Plex Panel is designed for the quantitative determination of EGF Eotaxin FGF basic G CSF GM CSF HGF IFN y IL 1 IL 1RA IL 2 IL 4 IL 5 IL 6 IL 8 IL 10 IL 12 IL 15 IL 17 I TAC MCP 1 MDC MIF MIG MIP 1c MIP 1 RANTES TNF a and VEGF in serum plasma and tissue culture supernatant This 28 Plex Panel is not intended to be combined with other assays 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 analys
13. ecaaecaeeeeeeaeeaeeseees 29 iii Kit Contents and Storage Storage All components of the Cytokine Monkey Magnetic 28 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 Cytokine Monkey Magnetic 28 Plex Panel are listed below Reagents Provided 100 Test Kit Cytokine Monkey Magnetic 28 Plex Antibody Bead Solution 1X 2 5 mL x 1 vial contains 0 05 sodium azide Cytokine Monkey Magnetic 28 Plex Biotinylated Antibody 1 mL x 1 vial Concentrate 10X contains 0 196 sodium azide Cytokine Monkey 14 Plex Standard contains 0 196 sodium azide 2 vials Cytokine Monkey 15 Plex Standard contains 0 1 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 1 x 96 well plate 96 well Flat Bottom Plate 1 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 prot
14. eins in panels designed by the researcher The Cytokine Monkey Magnetic 28 Plex Panel contains all the reagents that are intended for use with the Luminex 100 200 or Flex MAP 3D dual laser detection system with xPONENT software These instruments are manufactured by Luminex Corporation and are 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 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 100 200 or FlexMAP 3D instrument which monitors th
15. ency 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 Check standard reconstitution and dilution as described on page 9 Continued on next page Troubleshooting Continued Problem Leaky filter plate Cause Solution remains on the bottom of the 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 of the 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 filter plate on the manifold surface 23 Appendix Technical Support World Wide Visit the Invitrogen website at www invitrogen com luminex 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 Support contact information e Access to the Invitrogen Online Catalog e Additional product information and
16. es 150 pL 150pL 150pL 150pL 150pL lf lt i NC OT N Blank 13 19 127 1 81 1 243 1729 L y lt U NV NV WM Ww Ww Ww Ww oY ov ov ov ov oY oY o o o oS o o GY Std2 Std3 Std4 Std5 Std6 Std7 Serum plasma 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 Continued on next page Preparing Reagents Continued Online Tool Go to http www invitrogen com luminex under Multiplex Solution Tools click Luminex Calculation Worksheet for auto calculation of all assay dilutions Preparing 1X Determine the number of wells required for the assay Antibody Beads The 28 Plex Antibody Beads are supplied as a 1X solution that is ready to use The fluorescent beads are light sensitive Protect antibody conjugated beads from light during handling 10 Assay Procedure Washing Methods This assay may be washed using a vacuum manifold requires the filter bottom plate provided or may be washed using the aid of a magnetic separator requires the flat bottom plate provided Incomplete washing adversely affects assay results Perform all wash steps with the Wash Solution supplied with the kit All phases of the assay including incubations washing steps and loading beads are performed in one of the plates supplied with the kit Filter Plate Method l To wash beads place
17. g documents Directs the user to consult instructions for use IFU accompanying the product Copyright Invitrogen Corporation 08 July 2010 29 30 ii 31 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. invitrogen ij j Y Cytokine Monkey Magnetic 28 Plex Panel For simultaneous quantitative determination of EGF Eotaxin FGF basic G CSF GM CSF HGF IFN y IL 1 IL 1RA IL 2 IL 4 IL 5 IL 6 IL 8 IL 10 IL 12 IL 15 IL 17 I TAC MCP 1 MDC MIF MIG MIP 1o MIP 18 RANTES TNF a and VEGF in Rhesus monkey and Cynomolgus monkey serum plasma and tissue culture supernatant Catalog no LPC0003M Rev 0 0 08 July 2010 PRLPC0003M Table of Contents Table of Contents 2 eodein eie redet sou deed tenet en ana iii Kit Contents and Storage io erue ia EE nes iv TEP OCUCUIO 3 baad aeriene eraoro E 1 eu TP ctl 1 Before Starting ceed iid eb dl o D T t ei a 4 Preparing Reagents 5 see e eb mc D HR Ree een 7 Assay PIOCeQUfe tee E EE OR PERS RE eR TREE CERIS 11 Instrument Setups tob iet eese eedem tU Ma ES 19 Performance Characteristics and Limitations of the Procedure 20 Troubleshooting 55 recep Heh Aenean nena Aeg Saadeh 21 Append o 24 Techni Cal Support aree ert Ee ER E ERBEN ER eee 24 Purchaser Notification eesssseeseeeseeee eerte enne eene trennen enne 25 Referenc s iioii c ea deat i be E de o e dr edle 26 Protocol Summary aba eee e e b ie debe eb eda beaediig 27 Plate Plan Template eene ete tette de nme dee etnia eee Terni 28 Explanation of Symbols c cccccecscsscceecssesecesecsecseeeecesecasecseeeecsa
19. is with reproducibility similar to ELISA This assay uses 6 5 um polystyrene beads which contain magnetite Assays performed with these beads may be washed using a filter plate washed manually with the aid of a magnetic separator or washed with the aid of automated magnetic bead washing equipment The beads 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 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
20. iting 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 25 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 favorable clinical markers of disease status J Exp Med 200 331 343 Pickering A et al 2004 Cytokine response to infe
21. justable precision pipettes preferably with disposable plastic tips a manifold multi channel pipette is desirable Distilled or deionized water Glass or polypropylene tubes Aluminum foil Continued on next page Before Starting Continued Procedural Notes Review the procedural notes below before starting the protocol All phases of the assay are performed using either the filter plate or flat bottom plate provided Do not invert the plates during the assay The filter plate is provided for use when washing steps are performed with a vacuum manifold Do not exceed 5 mm Hg With the filter bottom plate contents are emptied from the bottom of the plate during washing The flat bottom plate is provided for use when washing steps are performed with a magnetic separator With the flat bottom plate contents are removed from the top of the plate during washing Washing with the flat bottom plate may be performed manually or with the aid of automated washing equipment 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 tu
22. lth Administration when handling and disposing of infectious agents 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 and samples which may be run as a singlet or in duplicate as desired NOTE 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 Cytokine Monkey Magnetic 28 Plex Panel includes both antibody bead reagents and buffer reagents Prepare components of the Cytokine Monkey Magnetic 28 Plex Panel according to instructions below Note Bring all reagents and samples to room temperature before use Upon storage at 2 to 8
23. mple type is RPMI medium containing 596 FBS the standards should be reconstituted in a mixture composed of 50 Assay Diluent and 50 RPMI containing 5 FBS The impact of adding additional standards to this assay has not been evaluated To the standard vials add the suggested reconstitution volume of the appropriate diluent see 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 Two vials of standards The preparation of the 28 plex standard curve requires one Preparing Standard Curve 150 uL Reconstituted Standard Std1 vial of Cytokine Monkey 14 Plex Standard plus one vial of Cytokine Monkey 15 Plex Standard To prepare Standard 1 first reconstitute each vial with 0 5 mL of appropriate diluent Then combine 300 uL from each vial and mix by gently pipetting up and down 5 to 10 times The standard curve is made by serially diluting the reconstituted standard in Assay Diluent for serum and plasma samples or a mixture of 50 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 tim
24. nued Analyte Capture 14 Choose the filter bottom plate when washing with a vacuum manifold Choose the flat bottom plate when washing manually with a magnetic separator or with automated magnetic bead washing equipment An adhesive plate cover may be used to seal any unused wells this will keep the wells dry for future use The filter bottom plate requires pre wetting before use in the assay Pre wet the designated wells of the filter bottom plate by adding 200 uL of Working Wash Solution Incubate the plate 30 seconds at room temperature Aspirate the Working Wash Solution from the wells using the vacuum manifold The solid bottom plate may be used without this pre wetting step Vortex the 1X Antibody Bead Solution for 30 seconds then sonicate for at least 30 seconds immediately prior to use in the assay The magnetic beads settle rapidly It is therefore important that the 1X Antibody Bead Solution is well mixed immediately prior to use Pipette 25 uL of the 1X Antibody Bead Solution into each well Once the beads are added to the plate keep the plate protected from light Add 200 uL Working Wash Solution to the wells Allow the beads to soak for 15 to 30 seconds Wash the wells two times aspirating the Working Wash Solution at the end of each washing step When using the filter plate blot the bottom of the plate on clean paper towels to remove any residual liquid Note Place the filter plate on a plate cover or non a
25. observed 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 Cause Solution Magnetic bead settling Make sure that the rate of plate shaking is sufficient to keep the beads suspended during incubations and prior to analysis Bead aggregation 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 Loss of beads due to To prevent membrane tearing place the filter plate pipette tips on the side of the well rather membrane tearing 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 Clog in instrument or Remove probe sonicate for 5 minutes probe rinse the probe and reinstall Run an unclog protocol See instrument manual Probe height set Readjust the instrument probe height If incorrectly it is too low it could p
26. ract 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 10096 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 reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in wr
27. s Procedure Limitations 20 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 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 Samples are diluted in the assay 1 2 50 uL of sample and 50 uL of diluent relative to the standards Be sure to account for this dilution factor during sample calculations 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 aberrant 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 Problem During data analysis insufficient and or erratic bead count is
28. 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 ORE UK Tel Toll Free 1 800 955 6288 Tel 813 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail techsupport invitrogen com ipinfo invitrogen com eurotech invitrogen com MSDS Requests 24 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 conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding cont
29. 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 excess fluid from the bottom of the filter plate Do not invert plate Following the last aspiration and plate taps use a clean absor
30. tinylated Antibody Vol Biotin Diluent 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 15 Assay Procedure Continued Analyte Detection 16 After the 2 hour capture bead incubation remove the liquid from wells with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Wash the plate by adding 200 uL of Working Wash Solution to the wells Allow the beads to soak for 30 seconds Remove the liquid with the vacuum manifold filter bottom plate or with magnetic washing equipment flat bottom plate Repeat this washing step for a total of 2 washes The bottom of the filter plate should be blotted on clean paper towels to remove residual liquid after the second wash Add 100 uL of prepared 1X Biotinylated Detector Antibody page 15 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 100 200 or Flex MAP 3D instrument during this incubation step 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
31. 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 or plasma 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 Note Reconstituting Lyophilized Standards Each kit comes with 2 complete sets of standard vials so that 2 runs of the assay 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 with the Cytokine Monkey Magnetic 28 Plex Panel 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 or plasma 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 5096 5095 mixture For example When the sa
32. uncture the filter plate 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 21 Troubleshooting Continued Problem During washing steps the vacuum manifold does not aspirate the liquid from wells of the filter plate In house controls perform differently in subsequent assays 22 Cause The filter plate is clogged Lack of a tight seal Incorrect concentration entered in data analysis software Improper reconstitution or dilution of the standard Solution Dislodge the clog by gently pushing the pointed end ofa 15 mL plastic conical tube into the bottom of the plate under the clogged well This procedure clears the small opening 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 Hold the filter 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 The standard proteins included in Invitrogen s Bead Kits are calibrated to NIBSC preparations whenever possible This calibration assures lot to lot consist
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