RayBiotech, Inc. Quantibody Bovine Cytokine Array 2 --
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1. 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 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 carcin2. chip 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 l 2 hours Note Incomplete drying of slides before use may cause the formation of comet tails B Prepare Cytokine Standard Dilutions Note There is only one vial of standard provided in the two slide Kit which is enough for making two standard curves Reconstitute the lyophilized standard within one hour of usage If you must use the standard for two different days store only the Stdl dilution at 80 Prepare serial dilution of cytokine standards 100ul 100ul 100ul 100ul 100ul 100ul MY ASASAOWOWSO O Add 500ul Sample Diluent 200ul 200ul 200ul 200ul 200ul 2001 100ul Vial Labels Stdl 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 Stdl Quantibody Bovine Cytokine Array 2 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes A 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 stan
3. cross reactivity with other proteins The spiking recovery rate of the cytokines by the kit in 2x diluted bovine serum and conditioned media CM is listed in the following table The spiking recovery rate for bovine serum and culture media pg ml Spike in Serum Serum Ag Serum CM CM Ag CM aFGF 20 000 1094 4 24101 5 115 0 0 31207 8 156 bFGF 500 0 6 218 8 44 0 4 528 5 106 GASP 1 2 000 0 0 532 3 27 0 0 1306 5 65 IGF 1 20 000 13215 8 27889 9 73 0 0 15095 9 75 IL 2 20 000 0 0 11895 5 59 11 5 19158 4 96 IL 4 1 000 0 0 145 6 15 0 0 974 7 97 IL 15 20 000 375 5 11195 9 54 0 0 20552 1 103 MCP 1 20 000 117 7 4130 8 20 286 0 23316 7 115 NCAM 1 50 000 9202 8 25528 1 33 0 0 32485 6 65 VEGF 1 000 0 0 253 3 25 0 0 1416 8 142 Quantibody Bovine Cytokine Array 2 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 Control
4. 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 chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass chip in clean environment Because there is no barcode on the slide transcribe the slide serial number from the slide bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide is disassembled you might not have enough info to distinguish one slide from the other C Incubation Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or lt 70 ul of sample or reagent is used Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation Quantibody Bovine Cytokine Array 2 7 IV Protocol A Completely air dry the glass chip 1 Take out the glass
5. Function based selection THI TH2 TH17 Array QAH TH 1 QAH TH17 QAM TH17 e Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 e Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 e Chemokine Arrays QAH CHE 1 QAM CHE 1 e MMP Array QAH MMP 1 e Immunoglobin Isotype Array QAH ISO 1 QAM ISO G1 Cytokine Number based selection e 400 cytokines QAH CAA 9000 e 360 cytokines QAH CAA 8000 e 320 cytokines QAH CAA 7000 e 280 cytokines QAH CA A 6000 e 240 cytokines QAH CAA 5000 e 200 cytokines QAH CAA 4000 e 160 cytokines QAH CAA 3000 QAM CAA 3000 e 120 cytokines QAH CAA 2000 QAM CAA 2000 e 80 cytokines QAH CAA 1000 QAM CAA 1000 e 60 cytokines QAH ANG 1000 QAM CYT Q2000 e 40 cytokines QAH INF 3 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CYT 4 QAH CYT 5 QAH CYT 6 QAH CYT 7 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 e 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR CYT 3 QAM CHE 1 e 20 cytokines QAH CYT 1 QAH CYT 2 QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM INT 1 QAH TH17 1 QAM TH17 1 e 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 QAH ADI 1 QAM INT 2 QAR CYT 1 QAR CYT 2 QAR INF 1 QAN CYT 1 QAP CYT 1 QAH IGF 1 e less than 10 cytokines QAH ISO 1 QAH ADI 2 QAP CYT 1 QAM ISO GI Purpose based selection Custom Arrays e Choose from over 700 cytokine pool Any kind Any number e Order slide only or full service in house e Desired m
6. Quantibody Bovine Cytokine Array 2 Quantitative measurement of 10 Bovine cytokines Patent Pending Technology User Manual Version September 2014 Cat QAB CYT 2 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 ravbiotech com Email info raybiotech com Cytokine Detected FGF acidic aFGF FGF basic bFGF GASP 1 10 IGF 1 IL 2 IL 4 IL 15 MCP 1 NCAM 1 VEGF 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 Assay duration 6 hrs 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 See Section V For Array Map gt Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody Cytokine Capture antibod
7. arker not in our pool No problem For certain developmental fee we may be able to add the marker to your panel if the paired antibodies are available on the market Quantibody Bovine Cytokine Array 2 20 Check our website regularly for updated Quantibody products 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 2013 RayBiotech Inc Quantibody Bovine Cytokine Array 2 21 Quantibody Bovine Cytokine Array 2 22
8. dard 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 Longer incubation time is preferable for higher signals Note We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation if less than 70 ul of sample or reagent is used Note This step may be done overnight at 4 C for best results 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of Ix Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with H20 Quantibody Bovine Cytokine Array 2 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 t
9. ell and wash 5 times with 150 ul of 1x Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step F Fluorescence Detection 15 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side 16 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube add enough 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 Stdl 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 high
10. emperature 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 Ix Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer II with H O Note Incomplete removal of the wash buffer in each wash step may cause dark spots Background signal is higher than that of the spot D Incubation with detection antibody cocktail and wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 ul of the detection antibody cocktail to each well Incubate at room temperature for 1 2 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times with 150 ul of 1x Wash Buffer I and then 2 times with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step E Incubation with Cy3 equivalent dye Streptavidin and wash 12 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 13 Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for hour Quantibody Bovine Cytokine Array 2 10 14 Decant the samples from each w
11. er PMT for low signal cytokines and a low PMT for high signal cytokines Quantibody Bovine Cytokine Array 2 11 G Data Analysis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArrav 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 U Image scan laser scanner U Data extraction 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 GenePix etc 21 22 21 23 222 223 232 213 U 55 54 57 56 188 178 189 190 Data computation Q Analvzer U Final Result pg ml Quantibody Bovine Cytokine Array 2 12 V Cytokine Array Map Standard Curves POS1 POS2 aFGF bFGF GASP 1 IGF 1 IL 2 IL 4 IL 15 MCP 1 NCAM 1 VEGF QAB CYT 2 Standard Curves 1e 5 1e 4 1e 3 Signal Intensity 1e 2 1e 1 1e 1 1e 0 1e 1 1e 2 1e 3 1e 4 1e 5 1e 6 Concentration pg ml Quantibody Bovine Cytokine Array 2 13 VI 8 Point Standards After reconstitution of the lyophilized cytokine standard mix the 8 point cytokine concentration used for generating the standard curve of a given antigen is listed below The detection sensitivity of each protein in one experiment is user dependent Try our array specific Quantibody Q Analyzer to see your L
12. imit of Detection LOD Section VIII Serial standard concentration pg ml pg ml Control Std7 Std6 Std5 Std4 Std3 Std2 Std1 aFGF 0 55 165 494 1 481 4 444 13 333 40 000 bFGF 0 1 4 12 37 111 333 1 000 GASP 1 0 16 49 148 444 1 333 4 000 IGF 1 0 55 165 494 1 481 4 444 13 333 40 000 IL 2 0 55 165 494 1 481 4 444 13 333 40 000 IL 4 0 3 8 25 74 222 667 2 000 IL 15 0 55 165 494 1 481 4 444 13 333 40 000 MCP 1 0 55 165 494 1 481 4 444 13 333 40 000 NCAM 1 0 137 412 1 235 3 704 11 111 33 333 100 000 VEGF 0 3 8 25 74 222 667 2 000 Bovine Cytokine Array 2 Cross reactivity Test CAB DAB aFGF bFGF MCP 1 GASP 1 IGF 1 IL 15 IL 2 IL 4 NCAM 1 VEGF aFGF 11 677 284 203 282 350 329 294 299 303 329 bFGF 120 12128 36 35 65 51 34 39 42 50 MCP 1 420 420 18 713 423 457 446 384 459 401 439 GASP 1 118 37 39 37 886 72 47 48 72 62 80 IGF 1 42 13 8 20 8 863 7 6 46 9 16 IL 15 301 161 162 188 333 7167 233 234 206 236 IL 2 150 95 74 105 159 95 15 744 115 113 141 IL 4 228 126 97 147 161 135 149 14 465 165 153 NCAM 1 136 65 82 74 117 63 73 77 29 855 87 VEGF 184 95 86 121 163 233 127 130 129 11 554 Quantibody Bovine Cytokine Array 2 14 VII System Recovery The antibody pairs used in the kit have been tested to recognize their specific antigen Analysis of samples containing only a single recombinant protein found negligible
13. mined 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 400 human or 200 mouse cytokines in a single experiment This is not only one of the largest and most efficient arrays on the market for cytokine quantification but makes it more affordable for quantification of large number of proteins Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery Quantibody Bovine Cytokine Array 2 4 How It Works Array support MYYYY l 0 9 Incubation of Sample YYYY With arrayed antibody 1 2 hr Supports Cocktail of LANS 2 K KX Incubation with YYY Biotinylated Ab Labeled mama 4 streptavidin l l i i Incubation with YYY Cy3 equivalent dye l hr Labeled streptavidin ee Detection of signals M Samples l 2 hr Data analysis and graph Quantibody Bovine Cytokine Array 2 5 II Materials Provided Upon recei
14. olution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard reconstitution or Improper dilution Poor standard Reconstitute the lyophilized standard well at the room temperature before making serial dilutions Check pipettes and ensure proper curve serial dilutions Inadequate detection Increase laser power that the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use new cytokine standard vial for new set of experiment Discard any leftover Overexposure Lower the laser power Dark spots Completely remove wash buffer in each wash step High FE Insufficient wash Increase wash time and use more wash background buffer Dust Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Quantibody Bovine Cytokine Array 2 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
15. oma 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 Bovine Cytokine Array 2 18 y y y XI Experiment Record Form PMT Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 1 2 4 Std5 aj aj 3 Std4 6 Std3 5 6 7 Std2 8 Std J IE 11 L IL IE 14 15 16 Quantibody Bovine Cytokine Array 2 19 XII How to Choose Quantibody Products Species based selection e Human QAH e Mouse QAM e Rat QAR CYT 1 QAR CYT 2 QAR CYT 3 QAR CXT 4 QAR INF 1 e Porcine QAP CYT 1 QAP CYT 2 QAP CYT 3 QAP CYT 4 e Non Human Primates NHP QAN CYT 1 e Canine QAC CYT 1 QAC CYT 2 e Feline QAF CYT 1 e Bovine QAB CYT 1 QAB CYT 2
16. pt 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 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 Cy3equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Manual Te RR R RK N R 1 2 3 4 5 6 7 8 9 10 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Bovine Cytokine Array 2 6 HI General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contains cytokines We recommend the following parameters for other samples 50 to 100 ul of original or diluted serum plasma cell culture media or other body
17. s 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 Bovine Cytokine Array 2 16 IX Troubleshooting guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper dilution Check pipettes and ensure correct preparation Short incubation time Weak Signal Ensure sufficient incubation time and change sample incubation step to overnight Too low protein concentration in sample Don t make too low dilution or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Bubble formed during incubation Avoid bubble formation during incubation Atrays are not completed covered by Uneven signal reagent Completely cover arrays with s
18. simultaneously It combines the advantages of the high detection sensitivity and specificity of ELISA and the high throughput of multiplex arrays Like a traditional sandwich based ELISA it uses a pair of cytokine specific antibodies for detection A capture antibody is first bound to the glass surface After incubation with the sample the target cytokine is trapped on the solid surface A second biotin labeled detection antibody is then added which can recognize a different epitope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin labeled Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use array format which array Quantibody Bovine Cytokine Array 2 3 multiple cytokine specific capture antibodies onto a glass support allowing simultaneous detection of cytokines in one experiment In detail one standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody together with the positive controls is arrayed in quadruplicate The slide comes with a 16 well removable gasket which allows for the process of 16 samples in one slide Four slide chips can be nested into a tray which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously For cytokine quantification the array specific cytokine standards whose concentration has been predeter
19. y Glass Slide Support Quantibody Bovine Cytokine Array 2 I TABLE OF CONTENTS L HOV CL ACW sal a A 1 TNO GUC MOM L id sar fr dana l a 3 HOW LU W OLKS ia i iGO OO ne enacts 5 IH Materials Provided ii2 sce senuttin zzo foka 6 Additional Materials Required 6 III General Considerations nee 7 A Preparation of SampleS 22 111112 7 B Handling Glass Chips en 7 C cubao Moena ea a GG auton 7 IVe POLO GO l ge R 8 A Complete Air Dry the Glass Chip 8 B Prepare Cytokine Standard Dilutions 8 C Blocking and Incubation 122121 9 D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detection 1111112 11 G Data Analysis 0 eran W A 12 V Cytokine Array Map Standard Curves 13 VI 8 Point Standards used owocna no G lit 14 MIE System Recovery asa OGR AE 15 VIII Quantibody Q Analyzer cccccccececeeececeeseeeees 16 IX Troubleshooting Guide 17 X Select Quantibody Publications 18 XI Experimental Record Form 2222 121 19 XII How to Choose Quantibody Products tess CE 20 Quantibod 9 Bovine Cytokine Array 2 2 y y y I Introduction Cytokines play an important role in immunity apoptosis angiogenesis cell growth and differentiation The
20. y 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 recognized with an antibody that is linked to a protein conjugate or enzyme Detection of the enzyme complex can then be visualized through the use of a substrate that produces a detectable signal While the traditional method works well for a single protein the overall procedure is time consuming and requires a lot of sample With little sample to work with conservation of precious small quantities becomes a risky task With the advancements in microarray technology over the last decade more and more choices are available to scientists today A long standing leader in the field RayBiotech has pioneered the development of cytokine antibody arrays which have now been widely applied in the research community with hundreds of peer reviewed publications including JEM Cell and Nature Quantibody arrays Our quantitative array platform uses multiplexed sandwich ELISA based technology and enables researchers to accurately determine the concentration of multiple cytokines
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