RayBiotech, Inc. Quantibody Mouse Cytokine Antibody Array
Contents
1. Inadequate standard reconstitution or Improper dilution Poor standard curve Reconstitute the lyophilized standard at room temperature before making serial dilutions Check pipettes and ensure proper serial dilutions Inadequate detection Increase laser power so the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use a new cytokine standard vial for a new experiment Discard any leftovers Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High Insufficient wash Increase wash time and use more wash buffer background Dust Minimize dust in work environment before starting experiment Slide is allowed to dry out Take additional precautions to prevent slides from dying out during experiment Quantibody Mouse Cytokine Antibody Array 4000 19 Select Quantibody Publications Stechova et al Influence of Maternal Hyperglycaemia on Cord Blood Mononuclear Cells in Response to Diabetes associated Autoantigens Scandinavian Journal of Immunology 2009 70 2 149 158 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 El Karim et al Neuropeptides Regulate Expression o2. IL 10 Cytokine Array 5 40 IL 12p40 IL 13 IL 15 IL 17 IL 21 KC Leptin LIX MCP 1 MCP 5 M CSF MIG MIP la MIP 1v PF 4 RANTES TARC TCA 3 TNFa TNFRI TNFRII Quantibody Mouse Cytokine Array 6 40 4 1BB ACE ALK 1 CT 1 CD27 CD40L CTLA 4 Decorin Dkk 1 Dtk Endoglin Fey RIIB FIt 3 L Galectin 1 Galectin 3 Gas 1 Gas 6 GITR L HAI 1 HGF R IL 1 R4 IL 3 RB IL 9 JAM A Leptin R L Selectin Lymphotactin MadCAM 1 MFG E8 MIP 3B Neprilysin Pentraxin 3 RAGE TACI TREM 1 TROY TSLP TWEAK R VEGF RI VEGF R3 Quantibody Mouse Cvtokine Arrav 7 40 B7 1 BAFF R BTC C5a CCL6 CD48 CD6 Chemerin Clusterin CXCLI15 Cvstatin C DAN DLL4 EDAR Endocan Fetuin A H60 IL 33 IL 7 Ralpha Kremen 1 Limitin Lipocalin 2 LOX 1 Marapsin MBL 2 Meteorin Nope NOV Osteoactivin OX40 Ligand P Cadherin Periostin PIGF 2 Progranulin Prostasin Renin 1 Testican 3 TIM 1 TRAIL Tryptase epsilon Quantibody Mouse Cytokine Array 8 40 6Ckine Activin A ADAMTSI Adiponectin ANG 3 ANGPTL3 Artemin CCL28 CD36 Chordin CRP E Cadherin Epigen Epiregulin Fas Galectin 7 gp130 Granzyme B Gremlin IFN y R1 IL 17B IL 17B R IL 22 MIP 1B MMP 2 MMP 3 MMP 10 PDGF AA Persephin sFRP 3 Shh N SLAM TCK 1 TECK TGFB1 TRANCE TremL1 TWEAK VEGF B VEGF R2 One standard glass slide is spotted with 16 wells of identical Format cytokine antibody arrays Each antibody i
3. 333 1 000 Eotaxin 2 0 1 4 12 37 111 333 1 000 Fas L 0 14 41 123 370 1 111 3 333 10 000 G CSF 0 27 82 247 741 2 222 6 667 20 000 GM CSF 0 14 41 123 370 1 111 3 333 10 000 ICAM 1 0 14 41 123 370 1 111 3 333 10 000 IFNy 0 5 16 49 148 444 1 333 4 000 IL 10 0 3 8 25 74 222 667 2 000 IL 1B 0 5 16 49 148 444 1 333 4 000 IL 2 0 14 41 123 370 1 111 3 333 10 000 IL 3 0 3 8 25 74 222 667 2 000 IL 4 0 1 2 6 19 56 167 500 IL 5 0 14 41 123 370 1 111 3 333 10 000 IL 6 0 5 16 49 148 444 1 333 4 000 IL 7 0 14 41 123 370 1 111 3 333 10 000 IL 10 0 14 41 123 370 1 111 3 333 10 000 IL 12p40 0 1 4 12 37 111 333 1 000 IL 13 0 27 82 247 741 2 222 6 667 20 000 IL 15 0 137 412 1 235 3 704 11 111 33 333 100 000 IL 17 0 5 16 49 148 444 1 333 4 000 IL 21 0 27 82 247 741 2 222 6 667 20 000 KC 0 3 8 25 74 222 667 2 000 Leptin 0 137 412 1 235 3 704 11 111 33 333 100 000 LIX 0 27 82 247 741 2 222 6 667 20 000 MCP 1 0 5 16 49 148 444 1 333 4 000 MCP 5 0 1 4 12 37 111 333 1 000 M CSF 0 3 8 25 74 222 667 2 000 MIG 0 14 41 123 370 1 111 3 333 10 000 MIP 10 0 14 41 123 370 1 111 3 333 10 000 MIP 1y 0 1 4 12 37 111 333 1 000 PF 4 0 27 82 247 741 2 222 6 667 20 000 RANTES 0 5 16 49 148 444 1 333 4 000 TARC 0 5 16 49 148 444 1 333 4 000 TCA 3 0 3 8 25 74 222 667 2 000 TNE RI 0 1 2 6 19 56 167 500 TNE RII 0 3 8 25 74 222 667 2 000 TNFa 0 1 4 12 37 111 333 1 000 QAM CYT 6 Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Stdi 4 1BB 0 34 103 309 9
4. 444 13 333 40 000 TremL1 0 55 165 494 1 481 4 444 13 333 40 000 TWEAK 0 27 82 247 741 2 222 6 667 20 000 VEGF B 0 14 41 123 370 1 111 3 333 10 000 VEGF R2 0 14 41 123 370 1 111 3 333 10 000 Quantibody Mouse Cytokine Antibody Array 4000 17 VII System Recovery The antibody pairs used in the kits have been tested to recognize their specific antigen The spiking recovery rate of the cytokines by the kits in serum and cell culture media can be found in their individual manuals 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 Simplicity Easy to operate and requires no professional training With a simple copy and paste process the cytokine concentration is determined Outlier Marking amp Removing The software can automatically mark and remove the outlier spots for more accurate data analysis Normalization The program allows for intra and inter slide normalization for large number of samples Two Positive Controls The program takes the two positive controls in each array for normalization Two Analytical Algorithms Users can choose either linear regression or log log algorithms to meet their analytical needs Two Data Outputs standard curves and digital concentration User Intervention The program allows for user manual handling of those outlie
5. 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 isotope 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 By arraying multiple cytokine specific capture antibodies Quantibody Mouse Cytokine Antibody Array 4000 3 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 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 sam
6. 26 2 778 8 333 25 000 ACE 0 137 412 1 235 3 704 11 111 33 333 100 000 ALK 1 0 14 41 123 370 1 111 3 333 10 000 CT 1 0 55 165 494 1 481 4 444 13 333 40 000 CD27 0 34 103 309 926 2 778 8 333 25 000 CD40L 0 55 165 494 1 481 4 444 13 333 40 000 CTLA 4 0 3 10 31 93 278 833 2 500 Decorin 0 7 21 62 185 556 1 667 5 000 Dkk 1 0 55 165 494 1 481 4 444 13 333 40 000 Dtk 0 27 82 247 741 2 222 6 667 20 000 Endoglin 0 14 41 123 370 1 111 3 333 10 000 Fc RIIB 0 14 41 123 370 1 111 3 333 10 000 FIt 3L 0 34 103 309 926 2 778 8 333 25 000 Galectin 1 0 14 41 123 370 1 111 3 333 10 000 Quantibody Mouse Cytokine Antibody Array 4000 15 Galectin 3 0 3 8 25 74 222 667 2 000 Gas 1 0 3 8 25 74 222 667 2 000 Gas 6 0 3 10 31 93 278 833 2 500 GITRL 0 1 4 12 37 111 333 1 000 HAI 1 0 14 41 123 370 1 111 3 983 10 000 HGFR 0 34 103 309 926 2 778 8 338 25 000 IL 1 R4 0 55 165 494 1 481 4444 13 333 40 000 IL 3 RB 0 55 165 494 1 481 4 444 18 833 40 000 IL 9 0 27 82 247 741 2 222 6 667 20 000 JAM A 0 7 21 62 185 556 1 667 5 000 Leptin R 0 7 21 62 185 556 1 667 5 000 L Selectin 0 14 41 123 370 1 111 3 333 10 000 Lymphotactin 0 274 823 2 469 7 407 22 222 66 667 200 000 MadCAM 1 0 14 41 123 370 1 111 3 333 10 000 MFG E8 0 55 165 494 1 481 44
7. 44 13 333 40 000 MIP 3p 0 1 4 12 37 111 333 1 000 Neprilysin 0 27 82 247 741 2 222 6 667 20 000 Pentraxin 3 0 14 41 123 370 1 111 3 333 10 000 RAGE 0 34 103 309 926 2 778 8 338 25 000 TACI 0 69 206 617 1 852 5 556 16 667 50 000 TREM 1 0 14 41 123 370 1 111 3 333 10 000 TROY 0 5 16 49 148 444 1 333 4 000 TSLP 0 5 16 49 148 444 1 333 4 000 TWEAKR 0 34 103 309 926 2 778 8 338 25 000 VEGF R1 0 14 41 123 370 1 111 3 333 10 000 VEGF R3 0 14 41 123 370 1 111 3 983 10 000 QAM CYT 7 Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Stdi B7 1 0 5 16 49 148 444 1 333 4 000 BAFF R 0 1 4 12 37 111 333 1 000 BTC 0 3 8 25 74 222 667 2 000 C5a 0 1 4 12 37 111 333 1 000 CCL6 0 55 165 494 1481 4444 13 333 40 000 CD48 0 3 8 25 74 222 667 2 000 CD6 0 1 4 12 37 111 383 1 000 Chemerin 0 137 412 1 235 3 704 11 111 33 333 100 000 Clusterin 0 137 412 1 235 3 704 11 111 33 333 100 000 CXCL15 0 274 823 2 469 7 407 22 222 66 667 200 000 Cystatin C 0 3 8 25 74 222 667 2 000 DAN 0 137 412 1 2285 3 704 11 111 33 333 100 000 DLL4 0 55 165 494 1 481 4444 13 333 40 000 EDAR 0 27 82 247 741 2 222 6 667 20 000 Endocan 0 27 82 247 741 2 222 6 667 20 000 Fetuin A 0 137 412 1 2285 3 704 11 111 33 333 100 000 H60 0 3 8 25 74 222 667 2 000 IL 33 0 5 16 49 148 444 1 333 4 000 IL 7 Ra 0 55 165 494 1 481 4444 13 333 40 000 Kremen 1 0 5 1
8. 49 148 444 1 333 4 000 IL 17E 0 55 165 494 1 481 4 444 13 333 40 000 IL 17F 0 55 165 494 1 481 4 444 13 333 40 000 IL 1ra 0 5 16 49 148 444 1 333 4 000 IL 2 Ro 0 14 41 123 370 1 111 3 333 10 000 IL 20 0 27 82 247 741 2 222 6 667 20 000 IL 23 0 55 165 494 1 481 4 444 13 333 40 000 IL 28 0 3 8 25 74 222 667 2 000 I TAC 0 27 82 247 741 2 222 6 667 20 000 MDC 0 1 4 12 37 111 333 1 000 MIP 2 0 1 4 12 37 111 333 1 000 MIP 3a 0 1 4 12 37 111 333 1 000 OPN 0 27 82 247 741 2 222 6 667 20 000 OPG 0 27 82 247 741 2 222 6 667 20 000 Prolactin 0 14 41 123 370 1 111 3 333 10 000 Pro MMP 9 0 137 412 1 235 3 704 11 111 33 333 100 000 P selectin 0 5 16 49 148 444 1 333 4 000 Resistin 0 3 8 25 74 222 667 2 000 SCF 0 14 41 123 370 1 111 3 333 10 000 SDF 1a 0 137 412 1 235 3 704 11 111 33 333 100 000 TPO 0 137 412 1 235 3 704 11 111 33 333 100 000 VCAM 1 0 5 16 49 148 444 1 333 4 000 VEGF 0 5 16 49 148 444 1 333 4 000 VEGF D 0 5 16 49 148 444 1 333 4 000 QAM CYT 5 Serial standard concentration pg ml pg ml Cntri Std7 Std6 Std5 Std4 Std3 Std2 Stdi bFGF 0 7 21 62 185 556 1 667 5 000 BLC 0 14 41 123 370 1 111 3 333 10 000 Quantibody Mouse Cytokine Antibody Array 4000 14 CD30L 0 3 8 25 74 222 667 2 000 Eotaxin 0 1 4 12 37 111
9. 6 49 148 444 1 333 4 000 Limitin 0 1 4 12 37 111 333 1 000 Lipocalin 2 0 137 412 1 235 3 704 11 111 33 333 100 000 LOX 1 0 5 16 49 148 444 1 333 4 000 Marapsin 0 27 82 247 741 2 222 6 667 20 000 MBL 2 0 3 8 25 74 222 667 2 000 Meteorin 0 55 165 494 1481 4444 13 333 40 000 Nope 0 14 41 123 370 1 111 3 333 10 000 Quantibody Mouse Cytokine Antibody Array 4000 16 NOV 0 55 165 494 1 481 4 444 13 333 40 000 Osteoactivin 0 14 41 123 370 1 111 3 333 10 000 OX40 Ligand 0 5 16 49 148 444 1 333 4 000 P Cadherin 0 5 16 49 148 444 1 333 4 000 Periostin 0 5 16 49 148 444 1 333 4 000 PIGF 2 0 1 4 12 37 111 333 1 000 Progranulin 0 137 412 1 235 3 704 11 111 33 333 100 000 Prostasin 0 137 412 1 235 3 704 11 111 33 333 100 000 Renin 1 0 55 165 494 1 481 4 444 13 333 40 000 Testican 3 0 55 165 494 1 481 4 444 13 333 40 000 TIM 1 0 137 412 1 235 3 704 11 111 33 333 100 000 TRAIL 0 14 41 123 370 1 111 3 333 10 000 Tryptase 0 137 412 1 235 3 704 11 111 33 333 100 000 QAM CYT 6 Serial standard concentration pg ml pg ml Control Std7 Std6 Std5 Std4 Std3 Std2 Std1 6Ckine 0 27 82 247 741 2 222 6 667 20 000 Activin A 0 5 16 49 148 444 1 333 4 000 ADAMTS1 0 55 165 494 1 481 4 444 13 333 40 000 Adipone
10. 7 CNTRL Quantibody Mouse Cytokine Antibody Array 4000 8 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 Stdl 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 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 Be careful to use the corresponding cytokine standard for the matching glass slide 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 Co
11. Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation Molecular and Cellular Biology 2008 28 24 7414 7426 11 Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T lymphocytes FEBS Letters 2007 581 26 5087 5093 This reference validates mulitplex ELISA results for several analytes with standard ELISA test results 12 Piganelli et al Autoreactive T cell responses new technology in pursuit of an old nemesis Editorial Review Pediatric Diabetes 2007 8 249 251 Quantibody Mouse Cytokine Antibody Array 4000 20 XI Experiment Record Form PMT Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 EI 2 4 Std5 aj 4 5 Std4 6 Std3 5 6 7 Std2 8 Std LILI uim 11 NNNM d 14 15 16 Quantibody Mouse Cytokine Antibody Array 4000 21 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
12. Quantibody Mouse Cytokine Antibody Array 4000 Quantitative measurement of 200 mouse cytokines Patent Pending Technology User Manual Version Dec 2013 Quantibody Mouse Cytokine Antibody Array 4000 Combination of Quantibody Mouse Cytokine Array 4 Mouse Cytokine Array 5 Mouse Cytokine Array 6 Mouse Cytokine Array 7 and Mouse Cytokine Array 8 to quantitatively measure the concentration of 200 mouse cytokines Cat QAM CAA 4000 Quantibody Mouse Cytokine Array 4 Cat QAM CYT 4 Quantibody Mouse Cytokine Array 5 Cat QAM CYT 5 Quantibody Mouse Cytokine Array 6 Cat QAM CYT 6 Quantibody Mouse Cytokine Array 7 Cat QAM CYT 7 Quantibody Mouse Cytokine Array 8 Cat QAM CVT 8 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 Cytokine Detected 200 Axl CD27L CD30T CD40 CXCL16 EGF E selectin Fractalkine Quantibody Mouse GITR HGF IGFBP 2 IGFBP 3 IGFBP 5 IGFBP 6 IGF I IL 12p70 Cytokine Array 4 40 IL 17E IL 17F IL 1ra IL 2 Ra IL 20 IL 23 IL 28 I TAC MDC MIP 2 MIP 3o OPN OPG Prolactin Pro MMP 9 P selectin Resistin SCF SDF 1a TPO VCAM 1 VEGF VEGF D bFGF BLC CD30L Eotaxin Eotaxin 2 Fas L G CSF GM CSF Quantibody Mouse ICAM 1 IFNy IL 1a IL 1B IL 2 IL 3 IL 4 IL 5 IL 6 IL 7
13. able It gives visual output as well as digital values More information can be found in section VIII Quantibody Mouse Cytokine Antibody Array 4000 12 V Cytokine Array Map QAM CYT 6 1 2 3 4 5678 9101112 a POS2 4 1BB b ALK 1 CT 1 c CD40L CTLA 4 d Dkk 1 Dik e Fcg RIIB FIt 3L f Galectin 3 Gas 1 g GITR L HAIH h IL 1 R4 IL 3 Rb i JAM A Leptin R j Lymphotactin MadCAM 1 k MIP 3b Neprilysin l RAGE TACI m TROY TSLP n VEGF R1 VEGF R3 3 SS Sie SSS oq Go 0 OU 5 Sf a aie SS eG GO OU QAM CYT 4 1 2 3 4 5 6 7 8 SIO 1142 POS1 AR Axl CD30T CD40 EGF E selectin GITR HGF IGFBP 3 IGFBP 5 IGF I IL 12p70 IL 17F IL 1ra IL 20 IL 23 l TAC MDC MIP 3a OPN Prolactin Pro MMP 9 Resistin SCF TPO VCAM 1 VEGF D QAM CYT 7 1 2 3 4 5 6 7 8 9 10 11 12 POS1 POS2 B7 1 BAFF R BTC C5a CCL6 CD48 CD6 Chemerin Clusterin CXCL15 Cystatin C DAN DLL4 EDAR Endocan Fetuin A H60 IL 33 IL 7 Ra Kremen 1 Limitin Lipocalin 2 LOX1 Marapsin MBL 2 Meteorin Nope NOV Osteoactivin OX40 Ligand P Cadherin Periostin PIGF 2 Progranulin Prostasin Renin 1 Testican 3 TIM 1 TRAIL Tryptase 13 Quantibody Mouse Cytokine Antibody Array 4000 S53 xo 570 4 0 Q 0 Com 33 74 FQ 40 Q209 ODM QAM CYT 5 1 2 3 4 POS1 BLC Eotaxin 2 GM CSF IL 1a IL 3 IL 6 IL 12p40 IL 17 Leptin MCP 5 MIP 1a RANTES TNF RI 5 6 8 9 10 11 12 QAM CYT 8 1 2 3 4 5 8 7 8 98 70 11 12 POS1 POS2 6Ckine Ac
14. be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation Quantibody Mouse Cytokine Antibody Array 4000 7 IV Protocol READ ENTIRE PROTOCOL BEFORE STARTING Note There are nine sets of reagents for the nine different arrays Be careful to use the correct glass slide lyophilized cytokine standard and the detection antibody cocktail for the corresponding array The following is the procedure for processing any one of the arrays in the kit 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 at room temperature for another 1 2 hours Note Incomplete drying of slides before use may cause the formation of streaks or comet tails on a slide B Prepare Cytokine Standard Dilutions Note There is only one vial of standard provided in the two slide kit this 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 rc for future use Prepare serial dilution of cytokine standards 100u 100ul 100ul 100ul 100ul 100ul TS SOS TS CS Cy Add 500ul Sample Diluent 2001 20041 200ul 2001 200u 200u 100ul Vial Labels Stdi Std2 Std3 Std4 Std5 Std6 Std
15. ctin 0 14 41 123 370 1 111 3 333 10 000 ANG 3 0 55 165 494 1 481 4 444 13 333 40 000 ANGPTL3 0 137 412 1 235 3 704 11 111 33 333 100 000 Artemin 0 5 16 49 148 444 1 333 4 000 CCL28 0 137 412 1 235 3 704 11 111 33 333 100 000 CD36 0 274 823 2 469 7 407 22 222 66 667 200 000 Chordin 0 14 41 123 370 1 111 3 333 10 000 CRP 0 5 16 49 148 444 1 333 4 000 E Cadherin 0 14 41 123 370 1 111 3 333 10 000 Epigen 0 27 82 247 741 2 222 6 667 20 000 Epiregulin 0 274 823 2 469 7 407 22 222 66 667 200 000 Fas 0 14 41 123 370 1 111 3 333 10 000 Galectin 7 0 137 412 1 235 3 704 11 111 33 333 100 000 gp130 0 14 41 123 370 1 111 3 333 10 000 Granzyme B 0 27 82 247 741 2 222 6 667 20 000 Gremlin 0 137 412 1 235 3 704 11 111 33 333 100 000 IFNy R1 0 3 8 25 74 222 667 2 000 IL 17B 0 274 823 2 469 7 407 22 222 66 667 200 000 IL 17B R 0 137 412 1 235 3 704 11 111 33 333 100 000 IL 22 0 55 165 494 1 481 4 444 13 333 40 000 MIP 1B 0 5 16 49 148 444 1 333 4 000 MMP 2 0 27 82 247 741 2 222 6 667 20 000 MMP 3 0 14 41 123 370 1 111 3 333 10 000 MMP 10 0 1 4 12 37 111 333 1 000 PDGF AA 0 5 16 49 148 444 1 333 4 000 Persephin 0 5 16 49 148 444 1 333 4 000 sFRP 3 0 27 82 247 741 2 222 6 667 20 000 Shh N 0 14 41 123 370 1 111 3 333 10 000 SLAM 0 137 412 1 235 3 704 11 111 33 333 100 000 TCK 1 0 274 823 2 469 7 407 22 222 66 667 200 000 TECK 0 274 823 2 469 7 407 22 222 66 667 200 000 TGFB1 0 137 412 1 235 3 704 11 111 33 333 100 000 TRANCE 0 55 165 494 1 481 4
16. eostasis 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 a target protein is first 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 risky task To solve this problem take advantage of the innovations in microarray technology over the last decade A long standing leader in the field Ravbiotech 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 in Cell and Nature The Quantibody array Our quantitative array platform uses the multiplexed sandwich ELISA based technology and 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
17. f Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 Souqui re S et al T Cell tropism of simian T cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills Mandrillus sphinx J Med Primatol 2009 38 4 279 89 Sharma et al Induction of multiple pro inflammatory cytokines by respiratory viruses and reversal by standardized Echinacea a potent antiviral herbal extract Antiviral Research 2009 83 2 165 170 Altamirano Dimas et al Echinacea and anti inflammatory cytokine responses Results of a gene and protein array analysis Pharmacuetical Biology 2009 47 6 500 508 Cheung et al Cordysinocan a polysaccharide isolated from cultured Cordyceps activates immune responses in cultured T lymphocytes and macrophages Signaling cascade and induction of cytokines Journal of Ethonopharmacology 2009 124 1 61 68 Du et al P2 380 Identification and characterization of human autoantibodies that may be used for the treatment of prion diseases Alzheimer s and Dementia 2009 4 4 T484 T484 Van Rossum et al Granulocytosis and thrombocytosis in renal cell carcinoma a pro inflammatory cytokine response originating in the tumour Neth J Med 2009 67 5 191 4 10 Zhai et al Coordinated Changes in mRNA Turnover Translation and RNA Processing
18. n the slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x 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 shaking at room temperature for 5 minutes then decant Wash Buffer II Quantibody Mouse Cytokine Antibody Array 4000 11 17 Remove water droplets completely by one of the following ways e Put the glass slide into the Slide Washer Dryer and dry the glass slide by centrifuge at 1 000 rpm for 3 minutes without cap e Or dry the glass slide 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 If the signal intensity for different cytokines 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 with most of the microarray analysis software GenePix ScanArray Express ArrayVision or MicroVigene For quantitative data analysis our Quantibody Q Analyzer software is avail
19. ommended 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 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 slides 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 slide 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 70 ul of sample or reagent is used Several incubation steps such as step 7 blocking and incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may
20. on 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 l 2 hour Be careful to use the corresponding detection cocktail for the matching glass slide Note incubation may be done at 4 C for overnight Quantibody Mouse Cytokine Antibody Array 4000 10 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 1 hour 14 Decant the samples from each well and wash 5 times with 150 ul of Ix 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 i
21. ples 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 200 mouse or 100 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 Quantibody Mouse Cytokine Antibody Array 4000 4 How It Works Array support YYYYY IC Incubation of Sample FISS With arraved antibodv 1 2 hr Samples 9 Supports Cocktail of Biotin Ab RX KX t KA Incubation with fey T Biotinylated Ab Labeled 8 8 B B 4 streptavidin iq Incubation with vy Cy3 equivalent dye hr Labeled streptavidin Detection of signals i Data analysis and graph Quantibody Mouse Cytokine Antibody Array 4000 5 II Materials Provided Upon receipt all components of the Quantibody Array kit should be stored at 20 C At 20 C the kit will retain comple
22. rs and other analytical data Lower and Upper Limits Determination The program automatically marks out the values below or above the detection range Standard Deviation The program outputs the standard deviations of the quadruplicate spots for data accuracy Analytical Tips Q Analyzer analysis tips are included in the program Quantibody Mouse Cytokine Antibody Array 4000 18 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 or change sample incubation to an overnight step Too low protein concentration in sample Dilute starting sample less or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Bubble formed during incubation Handle and pipette solutions more gently De gas solutions prior to use Arrays are not completely covered by Uneven signal reagent Prepare more reagent and completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer between wells Comet tail formation Air dry the slide for at least 1 hour before usage
23. s arrayed 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 Quantibody Mouse Cytokine Antibody Array 4000 1 TABLE OF CONTENTS I OVE EVIOW A 1 Introduction 3 How It Works 5 IH Materials Provided 6 Additional Materials Regquired hh ss 6 III General Considerations e A hes 7 A Preparation of Samples s s hh he e hv hel 7 B Handling Glass Slides ee 7 C Incubation 7 Ve Protocol estet n b fu Er MUN RESO ants 8 A Complete Air Dry the Glass Slide 8 B Prepare Cytokine Standard Dilutions 8 C Blocking and Incubation 9 D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 11 F Fluorescence Detection eee 11 G Data Analysis restiterunt 12 V Cytokine Array Map 13 VI 8 Point Standards 14 V System RECOVETY ne i 18 VIII Quantibody Q AnalyZzer 18 IX Troubleshooting Guide 19 X Select Quantibody Publications hh sl 20 XI Experimental Record Form hh hh hh 2 Quantibody Mouse Cytokine Antibody Array 4000 2 I 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 hom
24. te activity for up to 6 months Once thawed the glass slide 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 _QAM CCA 4000 1 _ QAM CCA 4000 2 Quantibody Array Glass Slide 3 10 2 Sample Diluent 5 5 3 20X Wash Buffer I 10 15 4 20X Wash Buffer II 5 5 5 Lyophilized cytokine standard mix 5 10 6 Detection antibody cocktail 5 10 7 Cy3 equivalent dye conjugated 5 10 Streptavidin 8 Slide Washer Dryer 5 5 9 Adhesive device sealer 25 50 10 Manual 5 5 The independent sets of reagents for Quantibody Mouse Cytokine Array 4 5 6 7 and 8 were shipped in three different boxes Among all the reagents the glass slide lyophilized cytokine standard mix and detection antibody cocktail are array specific while all other reagents are interchangeable between the arrays Note See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil ddH O 1 5ml Polvpropvlene microcentrifuge tubes Quantibody Mouse Cytokine Antibody Array 4000 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 rec
25. 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 2013 RayBiotech Inc Quantibody Mouse Cytokine Antibody Array 4000 22
26. tivin A ADAMTSI Adiponectin ANG 3 ANGPTL3 Artemin CCL28 CD36 Chordin CRP E Cadherin Epigen Epiregulin Fas Galectin 7 gp130 Granzyme B Gremlin IFN y R1 IL 17B IL 17B R IL 22 MIP 1b MMP 2 MMP 3 MMP 10 PDGF AA Persephin sFRP 3 Shh N SLAM TCK 1 TECK TGFb1 TRANCE TremL1 TWEAK VEGF B VEGF R2 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 QAM CYT 4 Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Std1 AR 0 3 8 25 74 222 667 2 000 Axl 0 14 41 123 370 1 111 3 333 10 000 CD27L 0 27 82 247 741 2 222 6 667 20 000 CD30T 0 14 41 123 370 1 111 3 333 10 000 CD40 0 14 41 123 370 1 111 3 333 10 000 CXCL16 0 1 4 12 37 111 333 1 000 EGF 0 3 8 25 74 222 667 2 000 E selectin 0 5 16 49 148 444 1 333 4 000 Fractalkine 0 137 412 1 235 3 704 11 111 33 333 100 000 GITR 0 5 16 49 148 444 1 333 4 000 HGF 0 27 82 247 741 2 222 6 667 20 000 IGFBP 2 0 137 412 1 235 3 704 11 111 33 333 100 000 IGFBP 3 0 27 82 247 741 2 222 6 667 20 000 IGFBP 5 0 55 165 494 1 481 4 444 13 333 40 000 IGFBP 6 0 55 165 494 1 481 4 444 13 333 40 000 IGF I 0 14 41 123 370 1 111 3 333 10 000 IL 12p70 0 5 16
27. ver the incubation chamber with adhesive film during incubation if less than 70 ul of sample or reagent is used Quantibody Mouse Cytokine Antibody Array 4000 9 Note This step may be done overnight at 4 C for best results Longer incubation time is preferable for higher signal 8 Wash e Calculate the volumes of Wash Buffers required based on the number of samples being processed and the entire remaining protocol described below e Dilute 20x Wash Buffer I and 20x Wash Buffer II separately with ddH5O to generate the required volume of Ix Wash Buffer I and Ix Wash Buffer II For example 100 ul of 20x Wash Buffer I would be diluted to a final volume of 2 000 ul 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 after each wash step e Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1x Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min e Decant the 1x Wash Buffer I from each well wash 2 times 5 min each with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step 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 detecti
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