RayBiotech, Inc. Quantibody Porcine Cytokine Array 3
Contents
1. Species based selection e Human QAH e Mouse QAM e Rat QAR CYT 1 QAR CYT 2 QAR CYT 3 QAR CYT 4 QAR INF 1 e Porcine QAP CYT 1 QAP CYT 2 QAP CYT 3 QAP CYT 4 e Non Human Primates NHP QAN CYT 1 e Canine QAC CYT 1 QAC CYT 2 e Feline QAF CYT 1 e Bovine QAB CYT 1 QAB CYT 2 Function based selection e THI TH2 TH17 Array QAH TH 1 QAH TH17 QAM TH17 e Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 e Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 e Chemokine Arrays QAH CHE 1 QAM CHE 1 e MMP Array QAH MMP 1 e Immunoglobulin Isotype Array QAH ISO 1 QAM ISO G1 Cytokine Number based selection e 400 cytokines QAH CAA 9000 e 360 cytokines QAH CAA 8000 e 320 cytokines QAH CAA 7000 e 280 cytokines QAH CA A 6000 e 240 cytokines QAH CAA 5000 e 200 cytokines QAH CA A 4000 e 160 cytokines QAH CA A 3000 QAM CAA 3000 e 120 cytokines QAH CAA 2000 QAM CAA 2000 e 80 cytokines QAH CAA 1000 QAM CAA 1000 e 60 cytokines QAH ANG 1000 QAM CYT Q2000 e 40 cytokines QAH INF 3 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CYT 4 QAH CYT 5 QAH CYT 6 QAH CYT 7 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 e 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR CYT 3 QAM CHE 1 e 20 cytokines QAH CYT 1 QAH CYT 2 QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM INT 1 QAH TH17 1 QAM THI7 1 e 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 QAH ADI2. Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard reconstitution or Improper dilution Poor standard Reconstitute the lyophilized standard well at the room temperature before making serial dilutions Check pipettes and ensure proper curve serial dilutions Inadequate detection Increase laser power that the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use new cytokine standard vial for new set of experiment Discard any leftover Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High FE Insufficient wash Increase wash time and use more wash background buffer Dust Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Quantibody Porcine Cytokine Array 3 17 X Select Quantibodv Publications Quantibody products have been referenced in hundreds of publications Following are only some of the selections Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T lymphocytes FEBS Letters 200
3. 1 QAM INT 2 QAR CYT 1 QAR CYT 2 QAR INF 1 QAN CYT 1 QAP CYT 1 QAH IGF 1 e less than 10 cytokines QAH ISO 1 QAH ADI 2 QAP CYT 1 QAM ISO G1 Purpose based selection Custom Arrays e Choose from over 1000 cytokine pool Any kind Any number e Order slide only or full service in house e Desired marker not in our pool No problem For certain developmental fee we may be able to add the marker to your panel if the paired antibodies are available on the market Quantibody Porcine Cytokine Array 3 20 Note Quantibody is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price This product is for research use only 2014 RayBiotech Inc Quantibody Porcine Cytokine Array 3 21
4. antigen Analysis of samples containing only a single recombinant protein found no cross reactivity with other proteins The spiking recovery rate of the cytokines by the kit in 2x diluted porcine serum SR and conditioned media CM is listed in the following table pg ml Spike in SR SR Ag SR CM CM Ag CM Decorin 2 000 1 254 3 597 117 8 2 880 144 GASP 1 2 000 2 083 3 673 79 2 1 678 84 IGFBP 5 10 000 1 9 563 96 0 14 343 143 IL 15 20 000 193 8 229 40 0 27 794 139 IL 22 50 000 3 218 43 010 80 85 62 856 126 Insulin 50 000 115 18 735 37 86 60 971 122 IP 10 20 000 272 7 025 3496 0 27 244 136 MCP 1 500 114 271 3196 0 787 157 NCAM 1 50 000 65 251 111 795 93 0 48 171 96 TWEAK R 50 000 1 689 31 906 60 9 50 587 101 Quantibody Porcine Cytokine Array 3 15 VIII Quantibody Q Analyzer Quantibody Q Analyzer is an array specific Excel based program However it is not a simple calculation macro as it contains sophisticated data analysis 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 number of samples e Two Positive Controls The progr
5. myeloma patients Leukemia 2011 Mar 25 3 527 37 Shinmura D et al Pretreatment of Human Mesenchymal Stem Cells with Pioglitazone Improved Efficiency of Cardiomyogenic Transdifferentiation and Improved Cardiac Function Stem Cells 2011 Feb 29 2 357 66 Feng W et al A novel role for platelet secretion in angiogenesis mediating bone marrow derived cell mobilization and homing Blood 2011 Mar 2 Fleming J et al Probiotic helminth administration in relapsing remitting multiple sclerosis a phase 1 study Mult Scler 2011 Mar 3 Morelli T et al Angiogenic biomarkers and healing of living cellular constructs J Dent Res 2011 Apr 90 4 456 62 Christoph E et al Levels of VEGF but not VEGF165b are Increased in the Vitreous of Patients With Retinal Vein Occlusion Am J of Ophthalmology 2011 May 28 Lee A et al Bacterial and Salivary Biomarkers Predict the Gingival Inflammatory Profile J Periodontol 2011 May 12 Scanlon ST et al Airborne lipid antigens mobilize resident intravascular NKT cells to induce allergic airway inflammation J Exp Med 2011 Sep 19 Quantibody Porcine Cytokine Array 3 18 XI Experiment Record Form PMT Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 a 2 4 Std5 aj aj 5 Std4 6 Std3 5 6 7 Std2 8 Std J IL 11 L IL IL 14 15 16 Quantibody Porcine Cytokine Array 3 19 XII How to Choose Quantibody Products
6. the high detection sensitivity and specificity of ELISA and the high throughput of multiplex 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 labeled Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use array format which array Quantibody Porcine Cytokine Array 3 3 multiple cvtokine specific capture antibodies onto a glass support allowing simultaneous detection of cvtokines in one experiment In detail one standard glass slide is spotted with 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 in one slide Four slide chips can be nested into a tray which matches a standard microplate 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 c
7. 150 ul of 1x Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step F Fluorescence Detection 15 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 16 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube add enough Ix Wash Buffer I 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 II about 30 ml with gentle and gently shake at room temperature for 5 minutes 17 Remove water droplets completely by one of the following ways e Put the glass chip into the Slide Washer Dryer and dry the glass chip by centrifuge at 1 000 rpm for 3 minutes without cap e Or dry the glass chip by a compressed N stream e Or gently apply suction with a pipette to remove water droplets Do not touch the array only the sides 18 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength 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 Note 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 cyto
8. 7 581 26 5087 5093 Zhai et al Coordinated Changes in mRNA Turnover Translation and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation Molecular and Cellular Biology 2008 28 24 7414 7426 Toh HC et al Clinical Benefit of Allogeneic Melanoma Cell Lysate Pulsed Autologous Dendritic Cell Vaccine in MAGE Positive Colorectal Cancer Patients Clin Cancer Res 2009 15 7726 7736 Willingham SB et al NLRP3 NALP3 Cryopyrin facilitates in vivo caspase 1 activation necrosis and HMGBI release via inflammasome dependent and independent pathways J Immunol 2009 183 3 2008 15 Gujar SA et al Reovirus virotherapy overrides tumor antigen presentation evasion and promotes protective antitumor immunity Mol Cancer Ther 2010 9 11 2924 33 Rajkumar T et al Identification and validation of genes involved in gastric tumorigenesis Cancer Cell Int 2010 10 1 45 Lewis JA et al Analysis of Secreted Proteins as an in vitro Model for Discovery of Liver Toxicity Markers J Proteome Res 2010 9 11 5794 802 Montes AH et al The MMPI 16071G 2G single nucleotide polymorphism associates with the HAART related lipodystrophic syndrome AJDS 2010 24 16 2499 506 Yin JJ et al Cediranib AZD2171 Inhibits Bone and Brain Metastasis in a Preclinical Model of Advanced Prostate Cancer Cancer Res 2010 70 21 8662 73 Storti P et al HOXB7 expression by myeloma cells regulates their pro angiogenic properties in multiple
9. Quantibody Porcine Cytokine Array 3 Quantitative measurement of 10 porcine cytokines Patent Pending Technology User Manual Version Sept 2014 Cat QAP CYT 3 RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 _ Website www raybiotech com Email info raybiotech com Cvtokine Detected Decorin GASP 1 IGFBP 5 IL 15 IL 22 Insulin IP 10 10 MCP 1 NCAM 1 TWEAK R One standard glass slide is spotted with 16 wells of Format identical cvtokine antibodv arravs Each antibodv is arraved in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye Sample Volume 50 100 ul per array Reproducibility CV lt 20 Assay duration 6 hrs 20000000 20000000 00000000 00000000 00000000 20000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 20000000 00000000 00000000 20000000 00000000 See Section V For Array Map gt Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody Cytokine Capture antibody Glass Slide Support Quantibody Porcine Cyt
10. am takes 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 those 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 Quantibody Porcine Cytokine Array 3 16 IX Troubleshooting guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Short incubation time Weak Signal Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration in sample Don t make too low dilution or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Bubble formed during incubation Avoid bubble formation during incubation Atrays are not completed covered by Uneven signal reagent Completely cover arrays with solution
11. ay specific Quantibody Q Analyzer to see your Limit of Detection LOD Section VIII Serial standard concentration pg ml pg ml Control Std7 Std6 Std5 Std4 Std3 Std2 Std1 Decorin 0 5 16 49 148 444 1 333 4 000 GASP 1 0 5 16 49 148 444 1 333 4 000 IGFBP 5 0 55 165 494 1 481 4 444 13 333 40 000 IL 15 0 55 165 494 1 481 4 444 13 333 40 000 IL 22 0 137 412 1 235 3 704 11 111 33 333 100 000 Insulin 0 137 412 1 235 3 704 11 111 33 333 100 000 IP 10 0 55 165 494 1 481 4 444 13 333 40 000 MCP 1 0 1 4 12 37 111 333 1 000 NCAM 1 0 137 412 1 235 3 704 11 111 33 333 100 000 TWEAK R 0 137 412 1 235 3 704 11 111 33 333 100 000 Panel cross reactivity IGFBP NCAM TWEAK CAB DAB Decorin GASP 1 5 IL 15 IL 22 Insulin IP 10 MCP 1 1 R Decorin 58 073 30 23 128 126 83 111 121 59 15 GASP 1 11 65 398 20 119 99 69 131 133 60 9 IGFBP 5 13 11 3 283 103 95 81 33 144 59 18 IL 15 15 12 125 13 966 677 100 176 218 82 23 IL 22 6 7 7 72 5974 35 16 116 134 2 Insulin 18 57 105 178 152 52 551 128 121 97 29 IP 10 15 48 407 189 180 117 1 975 283 92 45 MCP 1 16 49 414 192 142 100 56 65 204 77 47 NCAM 1 11 63 26 163 128 85 140 155 52 092 17 TWEAK R 5 13 13 63 50 29 22 137 30 6 327 Quantibody Porcine Cytokine Array 3 14 VII Svstem Recoverv The antibody pairs used in the kit have been tested to recognize their specific
12. ent cell tvpes 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 array ELISA In this method target protein is first immobilized to a solid support The immobilized protein is then recognized with an antibody that is linked to a protein conjugate or enzyme Detection of the enzyme complex can then be visualized through the use of a substrate that produces a detectable signal While the traditional method works well for a single protein the overall procedure is time consuming and requires a lot of sample With little sample to work with conservation of precious small quantities becomes a risky task With the advancements in microarray technology over the last decade more and more choices are available to scientists today 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 JEM Cell and Nature Quantibody arrays our quantitative array platform uses multiplexed sandwich ELISA based technology and enables researchers to accurately determine the concentration of multiple cytokines simultaneously It combines the advantages of
13. for cell and tissue lysates If you experience high background or the readings exceed the detection range further dilution of your sample is recommended Handling glass chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass chip in clean environment Because there is no barcode on the slide transcribe the slide serial number from the slide bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide is disassembled you might not have enough info to distinguish one slide from the other C Incubation Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation 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 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 Quantibody Porcine Cytokine Array 3 J IV Protocol A Completelv air drv the glass chip 1 Take out the glass chip from the box and let it e
14. ing for 20 min e Decant the 1x Wash Buffer I from each well wash 2 times 5 min each with 150 ul of Ix Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer II with H O Note Incomplete removal of the wash buffer in each wash step may cause dark spots Background signal is higher than that of the spot D Incubation with detection antibody cocktail and wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 ul 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 with 150 ul of 1x Wash Buffer I and then 2 times with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step E Incubation with Cy3 equivalent dye Streptavidin and 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 hour Quantibody Porcine Cytokine Array 3 10 14 Decant the samples from each well and wash 5 times with
15. kines and a low PMT for high signal cytokines Quantibody Porcine Cytokine Array 3 11 G Data Analvsis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArrav Express ArrayVision or MicroVigene For quantitative data analysis our Quantibody Q Analyzer software is available It gives visual output as well as digital values More information can be found in section VIII Experiments U Image scan laser scanner U Data extraction EM 2 1 3 2 89 88 90 91 GenePix etc 21 22 21 23 222 223 232 213 55 54 57 56 188 178 189 190 Data computation Q Analyzer U Final Result pg ml Quantibody Porcine Cytokine Array 3 12 V Cytokine Array Map amp Standard Curves POSI POS2 Decorin GASP 1 IGFBP 5 IL 15 IL 22 Insulin IP 10 MCP 1 NCAM 1 TWEAK R QAP CYT 3 Standard Curves 1e 5 Decorin GASP 1 IGFBP 5 IL 15 IL 22 Insulin IP 10 MCP 1 NCAM 1 TWEAK R 1e 4 1e 3 Signal Intensitv 1e 2 1e 1 1e 1 1e 0 1e 1 1e 2 1e 3 1e 4 1e 5 1e 6 Concentration pg ml Quantibody Porcine Cytokine Array 3 13 VI 8 Point Standards After reconstitution of the lyophilized cytokine standard mix the 8 point cytokine concentration used for generating the standard curve of a given antigen is listed below The detection sensitivity of each protein in one experiment is user dependent Try our arr
16. okine Array 3 1 TABLE OF CONTENTS l HOV CL ACW sal a A 1 TNO GUC MOM L id sar fr dana l A vacate 3 HOW LU W OLKS ia i e tana ars ne enacts 5 IH Materials Provided ii2 sce senuttin aciiecinakoed 6 Additional Materials Required seen 6 MI General Considerations nee 7 A Preparation of Samples eee enenenennnza 7 B Handling Glass Chips en 7 CCU DAO t Gudea taneccne auton 7 IVe POLO GO l a A id 8 A Complete Air Dry the Glass Chip 8 B Prepare Cytokine Standard Dilutions 8 C Blocking and Incubation nn 9 D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detection c cee esee eee eens 11 G Data Analysis eine te E EA aes 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards nn nn 14 VII System Specificity ss iii ses be iii i teds seems neues stan 15 VIII Quantibody Q Analyzer ccccccceceeececeeeuseeeees 16 IX Troubleshooting Guide 17 X Select Quantibody Publications 000 seen 18 XI Experimental Record Form eee 19 XII How to Choose Quantibody Products tess ji seen ik 20 Quantibody Porcine Cytokine Array 3 2 I Introduction Cvtokines plav an important role in immunitv apoptosis angiogenesis cell growth and differentiation Thev are involved in interactions between differ
17. quilibrate 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 drv at room temperature for another l 2 hours Note Incomplete drying of slides before use may cause the formation of comet tails B Prepare Cytokine Standard Dilutions Note 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 Stdl dilution at 80 C Prepare serial dilution of cytokine standards 100ul 100ul 100ul 100ul 100ul 100ul A S03 OS Os 08 Add 500u1 Sample Diluent 200ul 200ul 200ul 200ul 200ul 200u1 100ul Vial Labels Stdi Std2 Std3 Std4 Std5 Std6 Std7 CNTRL 2 Reconstitute the Cytokine Standard Mix lyophilized by adding 500ul 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 Quantibody Porcine Cytokine Array 3 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes 4 Pipette 100u1 Stdl into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100ul Std2 to tube Std3 and so on 5 Add 100ul Sample Diluent to another tube labeled as CNTRL Do not add standard cytokines or samples to
18. rray kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass chip cytokine standard mix detection antibody cocktail and Cy3 equivalent dye conjugated Streptavidin should be kept at 20 C and all other components may be stored at 4 C The entire kit should be used within 6 months of purchase Components Item Description 1 Slide kit 2 Slide kit Quantibody Array Glass Chip Sample Diluent 20X Wash Buffer I 20X Wash Buffer II Lyophilized cytokine standard mix Detection antibody cocktail Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Manual Te RR Re RK N RK 1 2 3 4 5 6 7 8 9 10 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Porcine Cytokine Array 3 6 HI General Considerations A Preparation of Samples Use serum free conditioned media if possible If 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 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
19. the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Note Since the starting concentration of each cytokine is different the serial concentrations from Stdl to Std7 for each cytokine are varied which can be found in section VI C Blocking and Incubation 6 Add 100ul Sample Diluent into each well and incubate at room temperature for 30 min to block slides 7 Decant buffer from each well Add 100ul standard cytokines or samples to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals Note 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 if less than 70 ul of sample or reagent is used Note This step may be done overnight at 4 C for best results 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of Ix Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with H20 Quantibody Porcine Cytokine Array 3 9 e Optional for Cell and Tissue Lysates Put the glass chip with frame into a box with Ix Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shak
20. urve 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 400 human or 200 mouse cytokines in a single experiment This is not only one of the largest and most efficient arrays 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 Quantibody Porcine Cytokine Array 3 4 How It Works Array support YY VA l 0 9 Incubation of Sample FISS With arraved antibodv l 2 hr Supports Cocktail of kie a a d OY K KX Incubation with YYY Biotinylated Ab Labeled mama 4 streptavidin l l ig i Incubation with Yey Cy3 equivalent dye l hr Labeled streptavidin ee Detection of signals M Samples l 2 hr 4 Data analysis and graph Quantibody Porcine Cytokine Array 3 5 II Materials Provided Upon receipt all components of the Quantibody A
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