Manual - RayBiotech, Inc.
Contents
1. Buffer from each well wash 2 times 5 min each with 150 ul of 1X Wash Buffer Il at room temperature with gentle rocking Completely remove wash buffer in each wash step Dilute 20X Wash Buffer Il with H20 Incomplete removal of the wash buffer in each wash step may cause dark spots the background signals higher than the spots D Incubation with Biotinylated Antibody Cocktail amp Wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 yl of the detection antibody cocktail to each well Incubate at room temperature for 1 2 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer and then 2 times with 150 pl of 1x Wash Buffer Il at room temperature with gentle rocking Completely remove wash buffer in each wash step E Incubation with Cy3 Equivalent Dye Streptavidin amp Wash 12 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 13 Add 80 yl of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for 1 hour 14 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer at room temperature with gentle rocking Co2. Kelley PM Steele MM Hollingsworth MA Tempero RM Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution Ng LG ed PLoS ONE 2014 9 11 e112737 doi 10 1371 journal pone 01 12737 Species Mouse Sample Type Tissue Lysate Huang X Ren L Ye P Cheng C Wu J et al 2014 Peroxisome Proliferator Activated Receptor c Deficiency in T Cells Accelerates Chronic Rejection by Influencing the Differentiation of CD44 T Cells and Alternatively Activated Macrophages PLoS ONE 9 11 e112953 doi 10 1371 ournal pone 0112953 Species Mouse Sample Type Conditioned Media More citations for this product may be available Contact techsupport raybiotech com XV Experiment Record Form Date File Name Laser Power Dilution factor HAIGE EEE EI EI D XVI How to Choose a Quantibody9 Array Species based selection carne OAC SES Rab AL Function based selection Adhesion Molecule Angiogenesis Arrays Bone Metabolism Chemokine Arrays Arrays Arrays Custom Arrays Cytokine Arrays Growth Factor Arrays Folios IL 1 Family Arrays ae EECHER Inflammation Arrays Interleukin Arrays Isotyping Arrays MMP Arrays Obesity Arrays Ophthalmic Arrays Disease Receptor Arrays Th1 Th2 Th17 Arrays Cytokine Number based selection Arrays are available in the Quantibody platform to detect 660 human 200 mouse or 67 rat proteins GLP Compliant testing services are also avail
3. cytokines or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Since the starting concentration of each cytokine is different the serial concentrations from Std1 to Std7 for each cytokine are varied which can be found in Section X C Blocking amp Incubation 6 Add 100 ul Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides 7 Decant buffer from each well Add 100 ul standard cytokines or samples to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals This step may be done overnight at 4 We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation especially if less than 70 ul of sample or reagent is used 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of 1X Wash Buffer at room temperature with gentle rocking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer with H2O e Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1X Wash Buffer cover the whole glass slide and frame with Wash Buffer and wash at room temperature with gentle rocking for 20 min e Decant the 1x Wash
4. Quantibody Mouse Cytokine Array 1 Quantitative measurement of 20 mouse cytokines Catalog QAM CYT 1 User Manual Last revised May 2015 Caution Extraordinarily useful information enclosed K RayBiotech HR The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com D O e 0 Page 7 Additional Materials Required 3 3 9 TETT VI VII General Considerations A Sample Preparation B Handling Glass Slides C Incubation VIII Protocol A Completely Air Dry The Glass Slide B Prepare Cytokine Standard Dilutions C Blocking amp Incubation D Incubation with Biotinylated Antibody Cocktail amp Wash E Incubation with Cy3 Equivalent Dye Streptavidin amp Wash F Fluorescence Detection G Data Analysis Sen cocente ICT Ja Please read the entire manual carefully before starting your experiment En A En 2 Ada a 00 00 CO JN NN O Wu Co N OO X X X X 17 18 19 l 11 IV V VI X 2 I Overview GM CSF IFN gamma IL 1 alpha IL 1 F1 IL 1 beta IL 1 Cytokines F2 IL 10 IL 12 p70 IL 13 IL 17A IL 2 IL 3 IL 4 IL 5 IL 6 Detected IL 9 KC CXCL1 MCP 1 CCL2 M CSF RANTES CCL5 20 TNF alpha VEGF A See Section IX for Array Map One st
5. able To learn more about the Quantibody Antibody Array visit www RayBiotech com Quantibody Multiplex Elisa Array html Quantibody9 is the trademark of RayBiotech Inc This product is for research use only 2015 RayBiotech Inc 20
6. amp analysis IV Materials Provided Catalog Component Name 1 Slide Box gon DE Mouse Cytokine Array 1 Lyophilized QAM CYT 1 STD Standard Mix 1 Vial i d Mouse Cytokine Array 1 Biotinylated s QAM CYT 1B Antibody Cocktail 1 25 pl 2 x 1 25 pl Cy3 equivalent dye conjugated QA CY3E Streptavidin 5 pl 2x5 pl QA SWD Slide Washer Dryer 1 x 30 ml Tube oonan mese rim 4 slide kits are comprised of 2 separate 2 slide kits See Section X for detailed cytokine concentrations after reconstitution V Storage Upon receipt all components should be stored at 20 C The kit will retain activity for up to 6 months Once thawed the glass slide standard mix antibody cocktail and dye conjugated Streptavidin should be kept at 20 C All other components may be stored at 4 C The entire kit should be used within 6 months of purchase VI Additional Materials Required e Benchtop rocker or orbital rocker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5 ml Polypropylene microcentrifuge tubes VII General Considerations A Preparation of Samples e Use serum free conditioned media if possible e f serum containing conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contains cytokines e We recommend the following parameters for your samples 50 to 100 ul of original or diluted serum plasma cell culture media or
7. andard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Method list of compatible laser scanners Il Introduction Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in interactions between different cell types cellular responses to environmental conditions and maintenance of homeostasis In addition cytokines are also involved in most disease processes including cancer and cardiac diseases The traditional method for cytokine detection and quantification is through the use of an enzyme linked immunosorbent assay ELISA In this method target protein is immobilized to a solid support The immobilized protein is then complexed with an antibody that is linked to an enzyme Detection of the enzyme complex can then be visualized through the use of a substrate that produces a detectable signal While this traditional method works well for a single protein the overall procedure is time consuming and requires a relatively high volume of sample Thus conservation of precious small sample quantities becomes a challenging task Innovations in microarray technology over the last decade have addressed this problem A long standing leader in the field Raybiotech has pioneered the development of cytokine antibody arrays which have now been widely applied in the research community with hundreds of pe
8. ay Express ArrayVision MicroVigene etc GAL files can be found here www RayBiotech com Gal Files html Need help analyzing all that data Copy and paste your data into the Q Analyzer Tool specific for this array catalog number QAM CYT 1 SW More information can be found in Section XII Experiment E Image Scan A list of compatible laser scanners can be found here RayBiotech com Scanners 4 Data Extraction GenePix etc Data Computation Q Analyzer software sold separately see section VIII E Analyze Results pg ml concentrations IX Array Map amp Standard Curves Each antibody is printed in quadruplicate horizontally A B EE Drees _ KA a L 6 1 Hf ias O _ MCP 1 C DE IN Mouse Cytokine Standard Curves Density Value IU X Standard Concentrations After reconstitution the lyophilized cytokine standard mix contains the following concentrations for each antigen included Serial standard concentration p x cw pue un pur er ume ila Cntrl Std7 Std5 mM LAN ma RANTES TNFa VEGF UJ Ea aw CC Cu EIS leg leg DERLAR E _ c un di ia XI Spiking amp Recovery The antibody pairs used in the kit have been tested to recognize their specific antigen The spiking and recovery rates of each cytokine in 2x dil
9. ch matches a standard microplate footprint and allows for automated robotic high throughput process of 64 arrays simultaneously For cytokine quantification the array specific cytokine standards whose concentration has been predetermined are provided to generate a standard curve for each cytokine In a real experiment standard cytokines and samples will be assayed in each array simultaneously through a sandwich ELISA procedure By comparing signals from unknown samples to the standard curve the cytokine concentration in the samples will be determined Quantibody array kits have been confirmed to have similar detection sensitivity as traditional ELISA Our current high density Quantibody kits allow scientists to quantitatively determine the concentration of 660 human 200 mouse and 67 rat cytokines in a single experiment This is not only one of the most efficient products on the market for cytokine quantification but makes it more affordable for quantification of large number of proteins Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery Ill How It Works YYY k Antibody array support la Y Y Y glass slide Incubation of sample amp 6F 4 standard protein cocktail dl of 1 2 e Incubation with biotinylated antibody cocktail 3 y 1 2 hours _ aii Incubation with Cy3 equivalent dye labeled streptavidin 1 hour Scan and perform data extraction
10. er reviewed publications including top tier journals such as Cell and Nature 3 The Quantibody array our multiplexed sandwich ELISA based quantitative array platform enables researchers to accurately determine the concentration of multiple cytokines simultaneously It combines the advantages of the high detection sensitivity amp specificity of ELISA and the high throughput of arrays Like a traditional sandwich based ELISA it uses a pair of cytokine specific antibodies for detection A capture antibody is first bound to the glass surface After incubation with the sample the target cytokine is trapped on the solid surface A second biotin labeled detection antibody is then added which can recognize a different epitope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin conjugated Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use an array format By arraying multiple cytokine specific capture antibodies onto a glass support quantitative multiplex detection of cytokines in one experiment is made possible In detail one standard glass slide is divided into 16 wells of identical cytokine antibody arrays Each antibody together with the positive controls is arrayed in quadruplicate The slide comes with a 16 well removable gasket which allows for the process of 16 samples on one slide Four slides can be nested into a tray whi
11. h array for normalization e Two Analytical Algorithms Users can choose either linear regression or log log algorithms to meet their analytical needs e Two Data Outputs standard curves and digital concentration e User Intervention The program allows for user manual handling of outliers and other analytical data e Lower and Upper Limits Determination The program automatically marks out the values below or above the detection range e Standard Deviation The program outputs the standard deviations of the quadruplicate spots for data accuracy e Analytical Tips Q Analyzer analysis tips are included in the program XIII Troubleshooting Guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper Check pipettes and ensure correct preparation dilution e SES Increase incubation time or change sample Weak Signal Short incubation time inc balion sicodo evemtight Too low protein Lessen dilution or do not dilute sample concentration in sample Concentrate sample if necessary a OF at Store kit as suggested temperature Don t prop 9 freeze thaw the slide Bubble formed during Decrease amount of rocking during incubations incubation check for bubble formation and remove bubbles Arrays are not Uneven signal completed covered by reagent Cover the incubation chamber with adhesive film Reagent evaporation SAN dur
12. ing incubation Cross contamination Avoid overflowing wash buffer and other solutions from neighboring wells into neighboring wells Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard Reconstitute the lyophilized standard well at the reconstitution or room temperature before making serial dilutions Improper dilution Check pipettes and ensure proper serial dilutions Completely cover arrays with solution for all required steps Increase laser power so the highest standard Inadequate detection concentration for each cytokine receives the highest possible reading yet remains unsaturated cytokine standards of experiment Discard any leftover SE STEEN Don t dry out slides during experiment XIV Publications Citing This Product 1 Huggenberger R et al Stimulation of lymphangiogenesis via VEGFR 3 inhibits chronic skin inflammation J Exp Med 2010 Sep 27 207 10 2255 69 doi 10 1084 jem 20100559 Species Mouse Sample Type Tissue Lysate 2 Kelly P et al Regressed lymphatic vessels develop during corneal repair Lab Invest 2011 Nov 91 11 1643 51 doi 10 1038 labinvest 201 1 121 Species Mouse 3 Guo H Yun C Hou G Du J Huang X et al Mangiferin Attenuates Th1 Th2 Cytokine Imbalance in an Ovalbumin Induced Asthmatic Mouse Model PLOS One Published June 23 2014 DOI 10 1371 journal pone 0100394 Species Mouse Sample Type Serum 4 Fink DM Connor AL
13. mpletely remove wash buffer in each wash step F Fluorescence Detection 15 16 17 18 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side Place the slide in the Slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x Wash Buffer about 30 ml to cover the whole slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer Wash with 1x Wash Buffer Il about 30 ml and gently shake at room temperature for 5 minutes Remove water droplets completely by gently applying suction with a pipette to remove water droplets Do not touch the array only the sides You may also dry the glass slide by a compressed N2 stream Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength green channel such as Axon GenePix Make sure that the signal from the well containing the highest standard concentration Std1 receives the highest possible reading yet remains unsaturated In case the signal intensity for different cytokine varies greatly in the same array we recommend using multiple scans with a higher PMT for low signal cytokines and a low PMT for high signal cytokines G Data Analysis 19 Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software GenePix ScanArr
14. other body fluid or 50 500 ug ml of protein for cell and tissue lysates If you experience high background or if the fluorescent signal intensities exceed the detection range further dilution of your sample is recommended B Handling Glass Slides e Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only e Handle all buffers and slides with powder free gloves Handle glass slide s in clean environment The Quantibody slides do not have bar codes To help distinguish one slide from another transcribe the slide serial number from the slide bag to the back of the slide with a fine point permanent marker Please write the number on the very bottom edge of the slide taking care to avoid writing on the array well areas C Incubation e Completely cover array area with sample or buffer during incubation e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rocking or rotation e Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or 70 ul of sample or reagent is used e Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation VIII Pro
15. tocol A Completely Air Dry The Glass Slide 1 Take out the glass slide from the box and let it equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry for another 1 2 hours Incomplete drying of slides before use may cause the formation of comet tails thin directional smearing of antibody spots B Prepare Cytokine Standard Dilutions There is only one vial of standard provided in the two slide kit which is enough for making two standard curves Reconstitute the lyophilized standard within one hour of usage you must use the standard for two different days store only the 5101 dilution at 80 C 100ul 100ul 100ul 100ul 100u1 100ul GS CX LC SS TSO TY CY Add 5001 Sample Diluent 20011 20011 200ul 20011 200u 200u 100ul Vial Labels 541 Std2 Std3 Std4 505 Std6 Std7 CNTRL 2 Reconstitute the Cytokine Standard Mix lyophilized by adding 500 ul Sample Diluent to the tube For best recovery always quick spin vial prior to opening Dissolve the powder thoroughly by a gentle mix Labeled the tube as Std1 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200 ul Sample Diluent to each of the tubes 4 Pipette 100 ul Std1 into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100 ul Std2 to tube Std3 and so on 5 Add 100 ul Sample Diluent to another tube labeled as CNTRL Do not add standard
16. uted serum SR plasma EDTA PLE plasma citrate PLC plasma heparin PLH and culture media CM are listed in the following tables The spiking recovery rate for culture media and serum Sole T seating ss cue Sem Deep Sensi Eo Lx wwe o o we EN RE IL 2 3 927 79 Ls mc 101 WIER NEL 1E 3L 28 2E ma mmc ero Sem ELE NE use o moe wow ECL ANGE JL WES SELLE INONE ER 3 war m ve lw lm pe remm Cam Y Dx esp we nxe Cer mp mos o we 20860 Ce XII Quantibody Q Analyzer The Q Analyzer is an array specific Excel based program It is much more than a simple calculation macro it performs sophisticated data analysis see below for description The Q Analyzer Tool specific for this array is catalog number QAM CYT 1 SW Key features e Simplicity Easy to operate and requires no professional training With a simple copy and paste process the cytokine concentration is determined e Outlier Marking amp Removing The software can automatically mark and remove the outlier spots for more accurate data analysis e Normalization The program allows for intra and inter slide normalization for large numbers of samples e Two Positive Controls The program utilizes the two positive controls in eac
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