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1. 5000 LeptinR_ 0 7 23 62 185 556 1 667 5000 L Selectin 0 14 a 1233 370 1111 3333 10 000 Lymphotactin O 274 823 2469 7 407 22222 66 667 200 000 MadCAM 1 O 14 41 123 370 1111 3333 10 000 MFGE8 0 55 165 494 1481 4444 13 333 40 000 MIP 3p 0 1 4 12 94 12 Neprilysin 0 27 82 247 741 2222 6667 20 000 Pentraxin3 0 14 a 123 370 1111 3333 10 000 RAGE O 34 103 309 926 2778 8333 25 000 Taci o0 69 206 617 1852 5556 16 667 50 000 O TREMA o 4 a 13 370 1111 3333 10 000 TROY 0o 5 16 49 148 444 1333 4000 TIP 0 5 16 49 148 444 1333 4000 TWEAKR 0 34 103 309 926 2778 8333 25 000 VEGFRi O 14 41 13 370 1111 3333 10 000 VEGFR3 0 14 41 123 370 1111 3333 10 000 Quantibody Mouse Cytokine Array 6 14 VII System Recovery The antibody pairs used in the kit have been tested to recognize their specific antigen The spiking recovery rate of the cytokines by the kit in 2x diluted mouse serum and 2x diluted mouse cell culture media CM is listed in the following table The spiking recovery rate for culture media and serum 4i1BB 10000 0 11573 116 420 7935 75 ACE 25 000 0 29636 119 36663 65869 117 ALK 1 5000 0 3
2. Analyzer to see your Limit of Detection LOD Section VIII Serial standard concentration pg ml 41BB Oo 34 103 309 926 2778 8333 25 000 ACE 0 187 42 1235 3704 11 111 33 333 100 000 Pp ALKA O 14 41 13 370 1111 3333 10 000 CTi 0o 165 494 1 481 4444 13 333 40000 CD27 0 34 103 309 926 2778 8333 25 000 cp4o o 5 165 494 1481 4444 13 333 40 000 CTLA4 0o 3 10 31 93 278 833 2 500 Decorin 0 7 2 Dki O 5 165 494 1481 4444 13 333 40 000 Dt o 277 82 247 741 2222 6667 20 000 Endogiin O 14 41 123 370 1111 3333 10 000 FeyRIB 0 14 a 123 370 1111 f 3333 10 000 FtsL O 34 103 309 926 2778 8333 25 000 Galectini 0 14 a 1233 370 1111 3333 10 000 Galectin 3 0 3 8 2 74 222 667 2000 Gat 0 K 3 amp 74 222 667 2000 1 Gas6 0 3 10 31 93 278 833 2500 GITRE o 1 4 12 37 m 33 1000 Hai 0o 14 a 123 370 1111 f 3333 10 000 HGFR o 34 103 309 926 2778 8333 25 000 h p wtR4 Oo 55 165 404 1481 4444 13 333 40000 IL BRB 0 55 165 494 1481 4444 13 333 40 000 L9 o 27 82 247 741 2222 6667 20000 JAMA 0 7 2t 62 185 556 1667
3. to dry out Don t dry out slides during experiment Poor standard curve Quantibody Mouse Cytokine Array 6 17 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 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 i
4. 396 68 140 2153 40 CT1 20 000 0 20353 102 655 9965 47 CD27 12500 0 143200 114 cbd40oL__ 20 000 O 18606 93 664 4877 21 CTLA 4 o o 319 64 4 154 30 Decorin 2500 0 3247 130 5 758 5378 Dkk 1 20 000 0 30416 152 10 527 30 558 100 Dtk 4000 0 1 350 135 88 1187 110 Endoglin 5 000 oO 4285 86 145 2645 50 FeyRIB 5000 Oo 3879 78 185 570 8 FItsL_ 12500 0 14418 115 1623 10565 72 Galectin 3 1000 0 1 254 125 1 004 1 508 50 Gas6 1 250 0 1130 90 1 528 2 226 56 GMRL_ 50 0 99 196 11 348 67 HA1 5000 0 4828 97 558 2546 40 HGFR 12500 O 11 060 88 302 4477 33 WiR4 20000 O 18350 92 48 2589 13 IL 3RB 20000 0 18 374 92 219 8464 41 ile 5000 0 3205 64 304 4723 88 JAMA 2500 0 1995 80 465 2313 74 LeptinR_ 2000 Oo 1648 82 593 1 523 47 L Selectin 5000 0 3709 74 13 561 14313 Lymphotactin 100 000 0 71279 71 1 238 35 303 34 MadCAM 1 5000 0 6682 134 127 2233 42 MFG E8 20000 0 27 037 135 3311 14198 54 MIP38 500 0 777 155 0 160 32 Neprilysin 10 000 0 10 444 104 1352 6 254 49 Pentr
5. Quantibody Mouse Cytokine Array 6 Quantitative measurement of 40 mouse cytokines Patent Pending Technology User Manual Version July 2010 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 4 1BB ACE ALK 1 CT 1 CD27 CD40L CTLA 4 Decorin Dkk 1 Dtk Endoglin Fcy RUB Flt 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 R1 VEGF R3 One standard glass slide is 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 Cytokine Detected 40 See Section V For Array Map gt Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody Cytokine Capture antibody Glass Slide Support Quantibody Mouse Cytokine Array 6 l TABLE OF CONTENTS le OVEVICW ae eee ere INtrOductiONn ccccccccccecessessseeceeceeeeseeasseeseeeeceeees How It Works 2 0 0 0 ccc ccc cece eee e cece eee e eee e
6. R CYT 2 QAR INF 1 QAH ISO 1 QAP CYT 1 Purpose based array Custom Arrays e Choose from over 400 cytokine pool Any kind Any number e Order slide only or full service in house Check our website regularly for updated Quantibody products Quantibody Mouse Cytokine Array 6 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 2010 RayBiotech Inc Quantibody Mouse Cytokine Array 6 2l
7. al 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 Array 6 16 IX Troubleshooting guide improper dilution 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
8. axin3 5 000 0 3143 63 820 13642 107 RAGE 12500 0 23478 188 0 14 948 120 TACI 25000 O 40802 163 375 19147 75 TREM 1 2500 0 2554 102 122 1270 46 TROY 2000 Oo 1665 83 280 1 062 30 TP 2000 O 1845 92 67 4 011 47 VEGFRi 5000 0 4786 96 1467 3 469 40 VEGFR3 5 000 o 5305 106 70 1549 30 O1 Quantibody Mouse Cytokine Array 6 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 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 analytic
9. ay 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 Kit 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 sy sy 2 3 4 5 6 7 8 9 pd en 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 Mouse Cytokine Array 6 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 tiss
10. be 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 1x 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 Mouse Cytokine Array 6 9 e Optional for Cell and Tissue Lysates Put the glass chip 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 mi
11. dards 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 160 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 Array 6 4 How It Works Array support YY YYY A Samples o Incubation of Sample YY YY With arrayed antibody 1 2 hr Supports Cocktail of KKK y KK K A Incubation with YYY Biotinylated Ab 1 2 hr Labeled E y streptavidin i ESES YYY VAA Detection of signals PA B Data analysis and graph Cy3 equivalent dye Incubation with 1 hr Labeled streptavidin Quantibody Mouse Cytokine Array 6 II Materials Provided Upon receipt all components of the Quantibody Arr
12. ee sse Il Materials Provided ccc ccc cc cence ecee eee ees Additional Materials Required 0oooooocmo II General Considerations 00 cc cceece eee e cece A Preparation of Samples ccceeeeee eens B Handling Glass Chips 00 c ee eee cece eee La ACUDAN rentar acces UV POOTO aai spree reporte peris A Complete Air Dry the Glass Chip B Prepare Cytokine Standard Dilutions O 0 0 0 N N JJ DO WD Ur QY ha C Blocking and Incubati0N ooooooocccccccc D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detecti0M oooooccococccccooorcccnooo 11 G Data AnalySis ccc ccc ccc cece cece eee ee rocas 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards 0 ccc cece cece cece nee e eee eens 14 VII System Recovery cece cece een e eee e nee e eee enees 15 VII Quantibody Q Analyzer oococcoccnccnccnccnccnccnnincnns 16 IX Troubleshooting Guide 0 ccc cece cece eens 17 X Select Quantibody Publications ccceeeeeeee 18 XI Experimental Record Form 0 cece eeeees 19 XII How to Choose Quantibody Products 20 Quantibody Mouse Cytokine Array 6 2 I Introduction Cytokines play an important role in innate immunity apopto
13. ermine 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 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 Quantibody Mouse Cytokine Array 6 3 specific capture antibodies onto a glass support multiplex detection of cytokines in one experiment is made possible 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 stan
14. mmune 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 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 Array 6 18 XI Experiment Record Form Date File Name Laser Power PMT Well No Sample Name CNTRL Std7 ER al E El 2 a a e Ed ele le lo Quantibody Mouse Cytokine Array 6 19 XII How to Choose Quantibody Product
15. n 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 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 lx 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 Quantibody Mouse Cytokine Array 6 10 14 Decant the samples from each well and wash 5 times with 150 ul of lx Wa
16. s Species based arrays e Human QAH TH 1 QAH INF 1 QAH INF 2 QAH INF 3 QAH CYT 1 QAH CYT 2 QAH MMP 1 QAH ISO 1 QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 QAH ADI 1 QAH ADI 2 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CAA 1000 QAH CAA 2000 QAH CAA 3000 QAH CAA 4000 QAH CAA 5000 QAH TH 17 e Mouse QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM CYT 4 QAM CYT 5 QAM CYT 6 QAM INF 1 QAM INT 1 QAM INT 2 QAM INT 1000 QAM CAA 1000 QAM CYT Q2000 QAM CAA 2000 QAM TH 17 e Rat QAR CYT 1 QAR CYT 2 QAR CYT 3 QAR INF 1 e Porcine QAP CYT 1 Function based arrays e TH1 TH2 TH17 Arrays QAH TH 1 QAH TH 17 QAM TH 17 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 MMP Array QAH MMP 1 e Immunoglobin Isotype Array QAH ISO 1 Cytokine Number based arrays e 240 cytokines QAH CAA 5000 e 200 cytokines QAH CAA 4000 e 160 cytokines QAH 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 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 QAH CYT 4 QAH CYT 5 e 20 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR CYT 3 e 20 cytokines QAH CYT 1 QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM INT 1 e 10 or less QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 QAH ADI 1 QAM INT 2 QAR CYT 1 QA
17. s and a low PMT for high signal cytokines Quantibody Mouse Cytokine Array 6 11 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 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 ee E y Data computation Q Analyzer y Final Result pg ml 55 54 57 56 188 178 189 190 Quantibody Mouse Cytokine Array 6 12 V Cytokine Array Map amp Standard Curves QAM CYT 6 Standard Curves 10 V O A gt 10 rat om 0 EJ LY tk i 10 4 ESA ELEY A 0 ay et 9h pra B ie aa a X C Ay E ond 7 S jE O REO AAA TA 0506 NE gt ais ZW V T y ZN LT AS 10 VEGF R3 101 107 10 101 10 109 104 10 Concentration pg ml Quantibody Mouse Cytokine Array 6 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 1s listed below The detection sensitivity of each protein in one experiment is user dependent Try our array specific Quantibody Q
18. sh 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 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 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 cytokine
19. sis angiogenesis cell growth and differentiation They are involved in interactions between different cell types cellular responses to environmental conditions and maintenance of homeostasis In addition cytokines are also involved in most disease processes including cancer and cardiac diseases The traditional method for cytokine detection and quantification is through the use of an enzyme linked immunosorbent array ELISA In this method 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 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 det
20. 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 There is only one vial of standard provided in the two slide kit which is enough for making two standard curves Reconstitute the lyophilized standard within one hour of usage If you must use the standard for two different days store only the Std1 dilution at 80 C Prepare serial dilution of cytokine standards 100ul 100u1 100ul 100u1 100ul 100ul OS SOS S amp S DY amp Add 500u1 Sample Diluent 200ul 200ul 200u1 200ul 200u1 20041 100u1 Vial Labels Std1 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 Mouse Cytokine Array 6 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes 4 Pipette 100u1 Std1 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 tu
21. ue 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 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 1s 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 Mouse Cytokine Array 6 7 IV Protocol A Completely air dry the glass chip 1 Take out the glass chip from the box and let it equilibrate to room

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