Manual
Contents
1. ccc ccc cc cence ecee eee ees Additional Materials Required c0cceees 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 Oy FIC D AION sacseeoe caeccne EET IVe POOTO aai E EE EE rcs eeeaas csaercees A Complete Air Dry the Glass Chip B Prepare Cytokine Standard Dilutions O o oo O N N N NOAOA NAA W e C Blocking and Incubation ccc eee D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detection cceeee cece ee ees 1 G Data AnalySis ccc ccc ccc cece cece eee ee eee eeaaees 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards 0 ccc cece cece cece nee e eee eens 14 VIL System Recovery cece cece een e eee e nee e eee enees 15 VIII Quantibody Q Analyzer cccecceseeseeseueeeeen 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 08 20 Quantibody Mouse Cytokine Array 5 2 I Introduction Cytokines play an important role in innate immunity apoptosis2. 5950 119 916 4903 8 ICAM 1 5000 0 4428 89 979 58373 88 IFNy 3 000 0 3208 107 865 4787 131 ila 1 000 0 1118 112 50 734 68 wip 2000 0 2368 118 162 2667 125 IL 2 5000 0 6736 135 2302 7 597 106 3 1000 0 1189 119 92 1148 106 i4 20 0 343 137 171 40 9 wes 5000 0 6334 127 2820 8874 121 we 2000 0 2673 134 193 2429 112 w7 5000 0 7132 143 1 261 6621 107 L40 5000 0 6948 139 467 4222 75 IL 12p40 500 0 494 99 476 951 95 L43 10 000 0 11 876 119 o0 6041 60 15 50 000 0 57 373 115 1 934 31 734 60 lL47 3 000 0 3572 119 1 660 5182 117 IL 21 20 000 0 16 992 85 239 19 663 97 Leptin 60 000 0 78428 131 5404 72 113 111 Ux 10000 0 14 080 141 5134 14 294 92 McpP 1 2000 0 2024 101 137 1762 8 mep 5 50 0 530 106 278 733 91 MCSF 1 000 87 1187 110 25 69 67 MiG 5000 0 6913 138 1719 4947 65 MIP tco 8000 0 6119 76 254 10 608 129 MiP ty 500 0 1 046 209 7 055 6804 over PF 4 10000 O 17 229 172 28 782 19 957 over 0 2770 92 83 3198 104 TaRc 6000 Oo 8255
3. Section VIII Serial standard concentration pg ml bFGF 0o 7 23 6 185 556 1 667 5000 pic 0 14 CD30 0o 3 8 amp 8 5 74 222 667 2000 Eotaxin 0o 1 4 12 3 m 333 1000 Eotaxing 0 1 4 12 37 1m f 333 1 000 Fast o 14 a 13 370 1111 3333 10 000 GcsF 0 27 82 247 741 2222 6667 20 000 GM csF 0o 14 a 13 370 1111 3 333 10 000 N IcaMmi o 4 a 13 370 1111 3 333 10 000 IFNy 0 5 16 43 1 amp 8 444 1333 4000 Pp llia 0 3 8 2 74 222 67 2000 iag 0 5 16 49 1488 444 1333 4000 i2 o 4 a 13 370 1111 3 333 10 000 Pp i3 o 3 8 amp 5 74 222 667 2000 i4 o 1 2 6 9 i5 o 4 a 13 370 1111 3333 10 000 pie o 5 16 49 148 444 1333 4000 i7 0o 4 a 13 370 1111 3 333 10 000 tio 0 14 a 123 370 1111 f 3333 10 000 w t2pao o 1 4 132 3z m 33 1000 pwd 0o 27 82 247 741 2222 6 667 20 000 ias 0 f 1377 412 1235 3704 11 111 33 333 100 000 p wiz o 5 16 4 18 44 wet 0 27 82 247 741 2222 6667 20 000 ke 0o 3 8 amp 8 5 7 4 222 67 2000 Leptin 0 137 412 1235 3704 11 111 33 333 100 000 LX 0
4. 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 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 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 5 10 14 Decant the samples from each well and wash 5 times with 150 ul of lx Wash
5. 138 87 6921 114 TCA3 1 000 oO 990 99 66 1053 99 TNFRI 250 0 202 81 3765 3987 89 TNFR 1 000 0 1 270 127 3 033 4204 117 TNFa 50 o0 397 79 42 31i 62 Quantibody Mouse Cytokine Array 5 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 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
6. 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 QAR 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 w
7. 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 determin
8. 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 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 Array 5 4 How It Works Array support YY YYY A Samples Incubation of Sample yyy With arrayed antibody 12 hr Supports Cocktail ot KKK 4 KK K A Incubation with YYY Biotinylated Ab 1 2 hr Labeled Sees 4 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 5 II Materials Provided Upon receipt all components of the Quantibody Array k
9. meet o 5 16 49 148 444 1333 4000 McP 5 o 1 4 132 3z m f 333 1000 Mcs 0o 3 8 2 74 222 67 2000 o Mme 0o 4 41 123 370 1111 3333 10 000 MPa o0 14 a 13 370 1111 3 333 10 000 Pp MPiy 0o 1 4 2 3z m 33 1000 PF4 0o 27 82 247 741 2222 6667 20 000 RANTES 0 5 16 49 148 444 1333 4 000 K CO K K NO N 00 NO tarc 0o 5 16 49 148 444 1333 4000 Teas 0 3 8 2 74 222 667 2 000 TNFRE 0 1 2 tte 50 TNFR 0 3 8 amp 8 5 74 222 67 2000 Ma 0o 1 4 132 3z m 333 1000 Quantibody Mouse Cytokine Array 5 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 bFGF 2500 0 2853 114 584 2878 92 BLC 10000 Oo 9191 92 7 002 16642 96 _ D30L 1 000 0 97 92 0o 71 79 Eotaxin 1 000 0 729 73 376 1419 104 Eotaxin 2 500 0 464 93 43 354 62 Fast 5000 0 6420 128 339 4846 90 GCSF_ 10 000 0 16 001 160 1 955 9837 79 GM CSF 5 000 0
10. quadruplicate spots for data accuracy e Analytical Tips Q Analyzer analysis tips are included in the program Quantibody Mouse Cytokine Array 5 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 to dry out Don t dry out slides during experiment Poor standard curve Quantibody Mouse Cytokine Array 5 17 Select Quantibody Publications Stechova et al Influence of Maternal Hyperglyc
11. 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 5 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 min
12. 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 cytokines
13. Quantibody Mouse Cytokine Array 5 Quantitative measurement of 40 mouse cytokines Patent Pending Technology User Manual Version July 2010 Cat QAM CYT S5 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 bFGF BLC CD30L Eotaxin Eotaxin 2 Fas L G CSF GM CSF ICAM 1 IFNy IL lo IL 18 IL 2 IL Cytokine Detected 3 IL 4 IL 5 IL 6 IL 7 IL 10 IL 12p40 IL 13 IL 40 15 IL 17 IL 21 KC Leptin LIX MCP 1 MCP 5 M CSF MIG MIP 1a MIP 1y PF 4 RANTES TARC TCA 3 TNFa TNFRI TNFRII 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 eee 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 5 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 ee sse II Materials Provided
14. aemia 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 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 charact
15. and a low PMT for high signal cytokines Quantibody Mouse Cytokine Array 5 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 J Image scan laser scanner l Data extraction 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 GenePix etc ee E J Data computation Q Analyzer J Final Result pg ml 55 54 57 56 188 178 189 190 Quantibody Mouse Cytokine Array 5 12 V Cytokine Array Map amp Standard Curves bFGF IL 10 IL 12p40 IL 13 IL 15 TNF RII Quantibody Mouse Cytokine Array 5 Signal Intensity vs Cytokine Concentration 1e 5 1e 4 1e 3 signal Intensity IU 1e 2 1e 1 1e 0 1e 1 1e 2 1e 3 1e 4 1e 5 1e 6 Cytokine Concentration pg ml Quantibody Mouse Cytokine Array 5 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 Analyzer to see your Limit of Detection LOD
16. e 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 5 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 standards
17. ebsite reqularly for updated Quantibody products Quantibody Mouse Cytokine Array 5 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 5 2l
18. erization 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 5 18 XI Experiment Record Form Date File Name Laser Power PMT Well No Sample Name CNTRL Std7 a a E 7 a a a PIL AL Lhe ede dbs Quantibody Mouse Cytokine Array 5 19 XII How to Choose Quantibody Products 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
19. it 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 5m Polypropylene microcentrifuge tubes Quantibody Mouse Cytokine Array 5 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
20. 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 5 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 tem
21. perature 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 100ul 100ul 100ul 100ul 100ul OS SOS S amp S DY amp Add 500u1 Sample Diluent 200ul 200ul 200ul 200ul 200u1 200ul 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 5 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes 4 Pipette 100ul Std1 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
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