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1. O 0 0 0 N NNW QV tA W C Blocking and Incubation D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detecti0M oooooccococccccooorcccnooo 11 G Data AnalySIS ooooooocccccccnorcccnnnnno nenne 12 V Cytokine Array Map esses ee 13 VI 8 Point Standards oooooccccccccccnoncccnnnnnnnno eee eens 14 VII System Recovery esssssse 16 VIII Quantibody Q Analyzer eee 17 IX Troubleshooting Guide 0 ccc cece cece eens 18 X Select Quantibody Publications s 19 Xl Experimental Record Form 20 Quantibody Mouse Cytokine Antibody Array 2000 Z 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 homeostasis In addition cytokines are also involved in most disease processes including cancer and cardiac diseases The traditional method for cytokine detection and quantification 1s 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 complexed with an antibody that 1s linked to an enzyme D
2. HGFR 0 34 103 309 926 2778 8333 25000 ira 0 55 165 494 1481 4444 13333 40000 IL3RB 0 55 165 494 1 481 4444 13 33 40000 L9 0 27 82 247 741 2222 6 67 20 000 JAMA 0 7 2t 62 185 556 1667 500 LepinR 0 7 2t 62 185 556 1667 5000 LSelcin 0 14 4t 123 370 1111 3333 10000 Lymphotacin 0 274 823 2469 7407 22222 66 667 200 000 MaCAM1 0 14 4t 123 370 1111 3333 10000 MFGEB 0 55 165 494 1481 4444 13 333 40000 MP3B 0 1 4 172 37 m 333 100 Neprilysin 0 27 82 247 741 2222 6667 20 000 Penraxn3 0 14 4t 123 370 1111 3333 10000 RAGE 0 34 103 309 926 2778 8333 25000 Taci 0 69 206 9617 1852 5 56 16 667 50000 TREM1 O 14 a 123 370 1111 3333 10000 TROY 0 5 16 49 i48 444 1333 400 Tsp o 5 16 49 148 444 1333 4000 TWEAKR 0 34 103 309 926 2778 8333 25000 VEGFRi 0 14 4t 123 370 1111 3333 10000 vegFR3 0 14 41 123 370 1111 3333 10 000 NO 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 individu
3. 2222 6 67 20000 RANTES 0 5 16 49 148 444 1333 400 TARC 0 5 16 49 148 444 1333 4000 TCAS 0 3 8 25 74 22 667 200 TNFRE 0 1 2 6 139 56 1607 50 TNFR 0 3 8 25 74 22 667 200 TNFa 0 j 1 4 132 37 m 338 100 QAM CYT 6 Serial standard concentration pg ml 41B 0 34 103 309 926 2778 8333 25000 ACE 0 137 412 1235 3704 11111 33 333 100 000 ALKA 0 1 4 a 123 370 1111 3333 10 000 Quantibody Mouse Cytokine Antibody Array 2000 15 CT 1 0 55 165 494 1 481 4 444 13 333 40 000 CD27 0 34 103 309 926 2778 8333 25000 CDL 0 55 165 494 1481 4444 13 333 40000 CTLA4 0 3 10 31 93 278 833 2 500 Decoin 0 7 2 Dk 0 55 165 494 1481 4444 13 333 40 000 Dk 0 27 s8 24 741 2222 6 67 20000 Endogiin 0 14 41 123 370 1111 3333 10000 FyRIB 0 14 4t 123 370 1111 3333 10 000 Ft3L O 34 10 309 926 2778 8333 25000 Galetin i 0 14 41 4123 370 1111 3333 10000 Galectin 3 0 3 8 25 74 22 667 200 Gai 0 3 8 25 74 222 667 2000 Gase o0 3 0 1 3a 93 278 833 2500 GIRL Oo 1 4 172 37 m 333 1000 HAM 0 1 4 a 123 370 1111 3333 10 000
4. 24 741 2222 6667 20000 wes o 55 165 494 1481 4444 13 333 40000 wes 0 83 8 25 222 6 2 000 Tace 0 27 82 24 741 2222 6 67 20000 MDC 0 1 4 12 3 m 333 100 MP2 o 1 4 172 37 m 333 100 MP3 0 1 4 132 3 m 333 100 OPN 0 27 s8 24 741 2222 6667 20000 OPG o0 27 82 24 741 2222 6 67 20000 Prolactin 0 14 4t 123 370 1111 3333 10 000 ProMMP 9 0 137 412 1235 3704 11 111 33 333 100 000 P selectin 0 5 16 49 148 444 1333 400 Rei 0 3 8 25 74 22 667 2 000 SCcF 0 14 a 13 370 1111 3333 10000 SDFia 0 137 412 1235 3704 11111 33 333 100 000 TPO 0 137 412 1235 3704 11 111 33 333 100 000 VCAMi 0 5 16 49 148 444 1333 400 VEGF 0 5 16 49 148 444 1333 400 VEGFD 0 5 16 49 148 444 1333 400 N C Y D O N Quantibody Mouse Cytokine Antibody Array 2000 14 QAM CYT 5 Serial standard concentration pg ml OBEGE T 9 C T do dep j 5 J B5 j 26 11 aber 5000 1 BCE 0 14 a 123 370 1111 3333 10 000 cpoL 0 3 a8 25 74 22 667 200 Edan 0 1 4 132 37 m 333 100 Eotaxin 2 0 1 4 172 37 m 333 100 Fast 0 14 4t 123 370 1111 33
5. Mi2 MP3s i Leptin ux MCP3 j L Selectin Eympnotacin MadCAM T k OPN OPG Prolactin McP s McsF MIG k meG e8 MIP 3b Neprilysin Pro MMP 9 P selectin Resistin Mita MiPg PF 4 Pontraxin 3 RAGE TAG m Scr SDF ta TPO m RANTES TARC TCA3 m TREM3T TROY TSIP n VCAM 1 VEGF VEGF D n TNFRI TNFRII n TWEAKR VEGF R1 VEGF R3 eoeo0 00050500000 eN 8883888 000066660000 SIME oOo0ooeeeeoooo 000000000000 TUER mere 000000000000 000000001000 000000000000 a eene O00000000000 zu iens 000066660000 ESSA nn OOoooeeoeoe0000 00000000 00000000 SEHE E O00o0ooeeeeoooo 00000000 00000000 00000000 00000000 nm ne 000000000000 00000000 0000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 e Cy3 equivalent dye 00000000 00000000 00000000 00000000 00000000 ooooooooQ 00000000 00000000 ee e nti qe Biotin Streptavidin complex e 3322333 33522333 Detect antibody 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 e Cytokine He HM Sosesess 39999999 Capture antibody 00000000 00000000 00000000 00000000 00000000 00000000 e Glass Slide Support Quantibody Mouse Cytokine Antibody Array 2000 13 VI 8 Point Standards After reconstitution of the lyophilized cytokine standard mix the 8 point cytokine concentration used for generating the standard curve o
6. 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 1s for research use only 2010 RayBiotech Inc Quantibody Mouse Cytokine Antibody Array 2000 21
7. 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 comet tails B Prepare Cytokine Standard Dilutions Note Reconstitute the lyophilized standard within one hour of usage Prepare serial dilution of cytokine standards 100ul 100u1 100u 100u1 100ul 100ul GS f Xy f y f Xy Py OS gt sample Diluent 20011 200u1 200u1 200u1 200u 200u 100ul Vial Labels Std1 Std2 Std3 Stdd 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 Stdl Quantibody Mouse Cytokine Antibody Array 2000 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 100u1 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
8. 1n each wash step Dilute 20x Wash Buffer II with H5O 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 Be careful to use the corresponding detection cocktail for the matching glass slide Note incubation may be done at 4 C for overnight 11 Decant the samples from each well and wash 5 times with 150 ul of Ix 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 Quantibody Mouse Cytokine Antibody Array 2000 10 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 Flu
9. 33 10000 GCSE 0 27 s8 247 741 2222 6667 20 000 GMCSF o 14 4t 123 370 1111 3333 10000 ICAMi 0 14 a 123 370 1111 3333 10 000 FEN O 5 16 49 148 44 1333 400 laa 0 3 8 25 74 22 667 2 000 MdB o 5 16 49 148 44 1333 400 lle o 14 4t 123 370 1111 3333 10000 L3 0 3 8 25 74 22 667 200 a l4 o 1 2 6 19 56 1607 50 IS 0 14 dt 123 370 1111 3333 10 000 lle 0 5 16 49 148 44 1333 4000 17 o 14 a 13 370 1111 3333 10 000 Ito 0 134 a 123 370 1111 3333 10 000 l t2p40 o 1 4 172 37 m 333 1000 a3 0 27 8 247 741 2222 6667 20000 Is 0 137 412 1235 3704 11111 33 333 100 000 17 0 5 16 49 148 444 1333 400 I co 148 4 tes 0 27 82 247 741 2222 6 67 20 000 KC 0 3 8 25 74 22 667 200 Lepin 0 137 412 1235 3 704 11111 33 333 100 000 Ux 0 MPAA 0 5 16 49 1488 444 1333 400 MCP5 o 1 4 172 37 m 333 100 MCSF 0 3 8 25 74 22 667 200 MG 0 14 41 123 370 1111 3333 10 000 Eh Eh NO N 00 NO MP o O 14 4t 123 370 1111 3333 10 000 MP y 0 1 PF4 o 27 s8 24 741
10. 60 human or 120 mouse cytokines in a single experiment This 1s 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 2000 4 How It Works Array support YY YYY A Samples o Incubation of Sample Vy v YY With arrayed antibody 1 2 hr Supports Cocktail of PR y KK K A Incubation with YYY Biotinylated Ab 1 2 hr Labeled 8 H BH B y streptavidin i e YYY J ae Detection of signals lat Data analysis and graph Cy3 equivalent dye Incubation with 1 hr Labeled streptavidin Quantibody Mouse Cytokine Antibody Array 2000 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 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 2 Slide Quantibody Array Glass Chip sample Diluent 20X Wash Buffer I 20X Wash Buffer II Lyophilized cytokine standard mix Detection ant
11. MIG MIP 1a MIP 1y PF 4 RANTES TARC TCA 3 TNFa TNFRI TNFRII 4 1BB ACE ALK 1 CT 1 CD27 CD40L CTLA 4 Decorin Dkk 1 Dtk Endoglin Fey RIIB Flt 3 L Quantibody Galectin 1 Galectin 3 Gas 1 Gas 6 GITR L HAI 1 Mouse Cytokine HGF R IL 1 R4 IL 3 RB IL 9 JAM A Leptin R L Array 6 40 Selectin Lymphotactin MadCAM 1 MFG E8 MIP 3p Neprilysin Pentraxin 3 RAGE TACI TREM 1 TROY TSLP TWEAK R VEGF R1 VEGF R3 One standard glass slide 1s spotted with 16 wells of Format identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye sample Volume 50 100 ul per array Reproducibility CV lt 20 Quantibody Mouse Cytokine Antibody Array 2000 l TABLE OF CONTENTS I OVerVi e WoutbosentuRRASUP eee ere INtrOductiONn ccccccccccecessessseeceeceeeeseeasseeseeeeceeees How It Works 2 0 0 0 ccc ccc cece eee e cece eee e eee e ee sse IL Materials Provided ccc ccc cc cence ecee eee ees Additional Materials Required III General Considerations 00 cc cceece eee e cece A Preparation of Samples sussesss B Handling Glass Chips 00 c ee eee cece eee C ACUDAN rentar acces 135 POOTO aai spree reporte peris A Complete Air Dry the Glass Chip B Prepare Cytokine Standard Dilutions
12. Quantibody Mouse Cytokine Antibody Array 2000 Quantitative measurement of 120 Mouse cytokines Patent Pending Technology User Manual Version July 2010 Quantibody Mouse Cytokine Antibody Array 2000 Combination of Quantibody Mouse Cytokine Array 4 5 and 6 to quantitatively measure the concentration of 120 Mouse cytokines Cat QAM CAA 2000 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 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 120 Arrays Included Quantibody 9 Mouse Cytokine Array 4 5 and 6 Axl CD27L CD30T CD40 CXCL16 EGF E selectin Fractalkine GITR HGF IGFBP 2 IGFBP 3 Quantibody IGFBP 5 IGFBP 6 IGF I IL 12p70 IL 17E IL 17F Mouse Cytokine IL 1ra IL 2 Ra IL 20 IL 23 IL 28 I TAC MDC Array 4 40 MIP 2 MIP 3a OPN OPG Prolactin Pro MMP 9 P selectin Resistin SCF SDF la TPO VCAM 1 VEGF VEGF D bFGF BLC CD30L Eotaxin Eotaxin 2 Fas L G Quantibody CSF GM CSF ICAM 1 IFNy IL lo IL 18 IL 2 IL Mouse Cytokine 3 IL 4 IL 5 IL 6 IL 7 IL 10 IL 12p40 IL 13 IL Array 5 40 15 IL 17 IL 21 KC Leptin LIX MCP 1 MCP 5 M CSF
13. al manuals Quantibody Mouse Cytokine Antibody Array 2000 16 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 1s 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 program 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 Mouse Cytokine Antibody Array 2000 17 IX Troubleshooting guide improper dilutio
14. ata extraction can be done with most of the microarray analysis software GenePix ScanArray Express Array Vision 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 y Image scan laser scanner y Data extraction 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 GenePix etc E eect 55 54 57 56 188 178 189 190 y Data computation Q Analyzer y Final Result pg ml Quantibody Mouse Cytokine Antibody Array 2000 12 V Cytokine Array Map QAM CYT 4 QAM CYT 5 QAM CYT 6 1 2 3 4 3 6 468 9 10 11 12 1 2 3 4 3 6 7 8 9 10 11 12 1 2 3 4 3 6 7 8 gI TIZ a Posi Pos2 AR a Post Pos2 bFGF a Post Pose 4185 b Aa co27 coaor b BLC CD30L Eotaxn o ACE Aki cri c chao cxcLie EGF o Eotaxn2 FasL G CSF c CD27 CD40L CTLA4 d E selectin Fractalkine GITR 4 GM CSF ICAM 1 IFNg d Decor Deci Dk e HGF iGreP2 IGFBP 3 e ra iib m2 e Endogin Fog RIB Fi3L Lierees IGFBP 6 Gri tes tea es f Galeotin 1 Galectin 3 Gas Y g it tep7o te 1 17 E me w7 f mio g Gase GRAL HAM h kara 1L2Ra m20 n 1240 113 f ias n HGFR IL1R4 Ls Rb i es i28 rrac taz wi ko ika JAMA LeptinR j Mbc
15. by the edges only Handle all buffers and slides with latex free gloves Handle glass chip 1n clean environment Because there 1s 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 1s 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 1s more than 2 hours or 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 Mouse Cytokine Antibody Array 2000 7 IV Protocol Note There are three sets of reagents for three different arrays Be careful to use the glass chip lyophilized cytokine standard and the detection antibody cocktail for the same array Following is the procedure for processing any one of the arrays in the kit A Completely air dry the glass chip 1 Take out the glass chip from the box and let it equilibrate to
16. 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 1004 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 olass 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 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 Longer incubation time is preferable for higher signal 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 Quantibody Mouse Cytokine Antibody Array 2000 9 20 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 shaking for 20 min e Decant the Ix 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
17. etection 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 Take the advantage of advancement in microarray technology over the last decade more and more choices are available to the scientist today A long standing leader in the field Raybiotech has pioneered the development of cytokine antibody arrays which has now been widely applied in the research community with hundreds of peer reviewed publications such as in Cell and Nature 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 specificity of ELISA and the high throughput of the arrays Like a traditional sandwich based ELISA it uses a pair of cytokine specific antibodies for detection A capture antibody 1s first bound to the glass surface After incubation with the sample the target cytokine 1s trapped on the solid surface A second biotin labeled detection antibody 1s then added which can recognize a different isotope of the target cytokine The cytokine antibody biotin complex can t
18. f a given antigen 1s listed below The detection sensitivity of each protein 1n one experiment 1s user dependent Try our array specific Quantibody Q Analyzer to see your Limit of Detection LOD Section VIII OAM CYT 4 Serial standard concentration pg ml L AR 0 3 8 25 74 22 667 200 A 0 14 a 13 37 1111 3333 10 000 CDL 0 27 s8 247 741 2222 6667 20 000 CD30T 0 4 41 123 370 1111 3333 10 000 CD40 o 14 4t 123 2370 1111 3333 10 000 Cxclte o 1 4 172 37 m 333 100 EGF 0 3 8 25 74 22 667 200 E selectin 0 5 16 49 Fractalkine 0 137 412 1235 3 04 11 111 33 333 100 000 aR 0 5 16 49 148 44 1333 400 He o 27 27 82 247 741 2222 6667 20 000 IGFBP2 0 137 412 1235 3704 11111 33 333 100 000 IGFBP3 0 27 82 24 741 2222 6 67 20000 IGEBP5 0 55 165 494 1481 4444 13 333 40000 IGFBP6 0 55 165 494 1481 4444 13 333 40000 IGF 0 14 dt 123 370 1111 3333 10 000 I 12p70 0 5 16 49 148 444 1333 400 ive 0 55 165 494 1481 4444 13333 40000 di 0 5 165 494 1481 4444 13 333 40000 laa 0 5 16 49 148 444 1333 4000 Il2Ra 0 14 a 123 370 1111 3333 10 000 020 o 27 82
19. hen 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 Quantibody Mouse Cytokine Antibody Array 2000 3 specific capture antibodies onto a glass support multiplex detection of cytokines 1n one experiment 1s made possible In detail one standard glass slide 1s 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 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 1
20. ibody cocktail Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Manual ER E 2 3 4 5 6 7 8 9 ER c There are three independent sets of reagents for Quantibody Mouse Cytokine Array 4 Quantibody 9 Mouse Cytokine Array 5 and Quantibody Mouse Cytokine Array 6 Among all the reagents the glass chip lyophilized cytokine standard mix and detection antibody cocktail are array specific while all the other reagents are suitable for all the three arrays Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Mouse Cytokine Antibody Array 2000 6 II 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 for cell and tissue lysates If you experience high background or the readings exceed the detection range further dilution of your sample is recommended B Handling glass chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides
21. n preparation Weak Signal change sample incubation step to overnight sample sample freeze thaw the slide Bubble formed during incubation Avoid bubble formation during incubation Arrays are not completed covered by Completely cover arrays with solution Uneven signal reagent film during incubation neighboring wells usage Inadequate standard reconstitution or Reconstitute the lyophilized standard well at Improper dilution the room temperature before making serial dilutions Check pipettes and ensure proper 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 Hish wash step 5 Insufficient wash Increase wash time and use more wash background buffer Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Poor standard curve Quantibody Mouse Cytokine Antibody Array 2000 18 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 Cryopy
22. orescence 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 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 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 Quantibody 9 Mouse Cytokine Antibody Array 2000 11 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 cytokines and a low PMT for high signal cytokines G Data Analysis 19 D
23. rin 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 of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 Souquiere 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 thrombocyto
24. sis 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 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 2000 19 Xl Experiment Record Form Date File Name Laser Power PMT Well No sample Name CNTRL Std7 ER al E 7 E B uH HN Ed ele le lo Quantibody Mouse Cytokine Antibody Array 2000 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

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