Manual - RayBiotech, Inc.
Contents
1. 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 III How It Works Yyy Mea Antibody array support la Y Y Y glass slide Incubation of sample amp standard protein cocktail T of 1 2 O Incubation with biotinylated antibody cocktail x 3 1 2 hours ee aii Incubation with Cy3 equivalent dye labeled streptavidin 1 hour Scan and perform data extraction amp analysis IV Materials Provided Catalog Component Name 1 Slide Box gon Human TH1 TH2 Array 1 Lyophilized QA2. 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 QAH TH 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 each 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 Inadeq
3. 0 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 If you must use the standard for two different days store only the Std1 dilution at 80 TC 100ul 100ul 100u1 100u1 100u1 100ul GS TSOS TSO YD Add 500u1 Sample Diluent 20011 20011 2001 200ul 20011 200n1 100ul Vial Labels Stdl Std2 Std3 Std4 Std5 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 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 e
4. H TH 1 STD Standard Mix 1 Vial Human TH1 TH2 Array 1 Biotinylated 3 z el QAH TH 1B Antibody Cocktail 1 25 ul 2 x 1 25 ul Cy3 equivalent dye conjugated QA SWD Slide Washer Dryer 1 x 30 ml Tube oonan pares Fim 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 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 r
5. Q Analyzer Tool specific for this array catalog number QAH TH 1 SW More information can be found in Section XIl Experiment 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 l Analyze Results pg ml concentrations IX Array Map amp Standard Curves Each antibody is printed in quadruplicate horizontally Mtl sf Se cE aE aE ES A Post POS2 D IL 8 CXCL8 IL 10 El IL 13 GM CSF IFN gamma TNF alpha Concentration pgimI X Standard Concentrations After reconstitution the lyophilized cytokine standard mix contains the following concentrations for each antigen included Serial standard concentration pg ml o Oa r Racal anc Rana Nan nal nal a IL 2 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 diluted 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 human culture media and serum Sini Se O DAN I RI RL II QI VO I s SL O I y o p e IO y VE e LL A I I LO e I IL m e m XII Quantibody Q Analyzer The Q Analyzer is an array specific Excel based program It is
6. Quantibody Human TH1 TH2 Array 1 Quantitative measurement of 10 human cytokines Catalog QAH TH 1 User Manual Last revised May 2015 Caution Extraordinarily useful information enclosed K Ke RayBiotech 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 O O D a v Page 7 Additional Materials Required 3 3 5 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 ow esse Please read the entire manual carefully before starting your experiment pare di Ada 1 0000 NNNYN O o1 0 N OO IX X X X X 17 18 19 l Il IV V VI X 2 I Overview Cytokines GM CSF IFN gamma IL 10 IL 13 IL 2 IL 4 IL 5 IL 6 IL 8 Detected CXCL8 TNF alpha 10 See Section IX for Array Map One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody is arrayed in quadruplicat
7. ach 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 H20 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 Buffer from each well wash 2 times 5 min each with 150 pl of 1X Wash Buffer Il at room temperature with gentle rocking Completely remove wash buffer in each wash step Dilut
8. ange 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 lt 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 Protocol 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 3
9. dwich 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 which matches a standard microplate footprint and allows for automated robotic high throughput process of 64 arrays simultaneously
10. e 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 peer reviewed publications including top tier journals such as Cell and Nature The Quantibody array our multiplexed san
11. e 20X Wash Buffer Il with H2O 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 pl 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 ul 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 ul 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 Completely 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 Caref
12. ons Citing This Product 1 Gao QT Cheung JKH Li J Jiang ZY Chu GKY et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal related kinase in cultured T lymphocytes FEBS Lett 2007 581 5087 5099 Species Human Sample Type Conditioned Media 2 Veiga P Schroth R Guedes R Freire S Nogueira Filho G Serum cytokine profile among Brazilian children of African descent with periodontal inflammation and sickle cell anaemia Volume 58 Issue 5 May 2013 Pages 505 510 Species Human Sample Type Serum 3 Chia C Kumari U Casey P Breast cancer cell invasion mediated by Galpha12 signaling involves expression of interleukins 6 and 8 and matrix metalloproteinase 2 Journal of Molecular Signaling 2014 9 6 doi 10 1186 1750 2187 9 6 Species Human Sample Type Conditioned Media 4 Epanchintsev A Pavithra S Verma R Lyakhovich A IL 6 IL 8 MMP 2 MMP 9 are overexpressed in Fanconi anemia cells through a NF B TNF alpha dependent mechanism Species Human 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 e e HE ei ee lls oO pei ar O XVI How to Choose a Quantibody Array Species based selection carne OAC Egune OAE Rab AL Function based selection Adhesion Molecule Angiogenesis Arrays Bone Metabolism Chemokine Ar
13. rays Arrays Arrays Custom Arrays Cytokine Arrays Growth Factor Arrays e IL 1 Family Arrays ae mespopee Inflammation Arrays Interleukin Arrays Isotyping Arrays MMP Arrays Obesity Arrays Ophthalmic Arrays oo 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 available To learn more about the Quantibody Antibody Array visit www RayBiotech com Quantibody Multiplex Elisa Array html Quantibody is the trademark of RayBiotech Inc This product is for research use only 2015 RayBiotech Inc 20
14. uate reagent volumes or improper Check pipettes and ensure correct preparation dilution ee Increase incubation time or change sample Weak Signal Short incubation time incubation step 19 everiaht Too low protein Lessen dilution or do not dilute sample concentration in sample Concentrate sample if necessary THES GOERS 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 SIAE during 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 A ie alowed Ioar Don t dry out slides during experiment XIV Publicati
15. ully 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 I 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 alow 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 ScanArray 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
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