RayBiotech, Inc. Quantibody Rabbit Cytokine Array 1
Contents
1. 10 063 38 TNFalpha 2 1 1 17 12 3 1 2 37 6 142 The spiking recovery rate for Rabbit serum and culture media CM pg ml Spike in CM CM Ag CM Serum Serum Ag Serum IL 1alpha 5000 0 5 1 3394 0 68 22 7 3233 7 64 IL 1beta 500 0 0 0 264 5 53 0 0 467 3 93 IL 8 100 0 0 3 109 4 109 279 5 309 4 30 IL 17A 10000 0 0 0 6280 7 63 0 0 8891 6 89 IL 21 200000 0 0 0 206523 0 103 5539 4 180660 5 88 Leptin 50000 0 0 0 42525 0 85 419 3 62977 8 125 MIP 1beta 200 0 2 1 252 7 125 11 0 153 4 71 MMP 9 5000 0 0 0 2432 7 49 1188 9 6801 0 112 NCAM 1 10000 0 0 0 3808 2 38 64948 3 77658 1 127 TNFalpha 2000 0 0 0 1354 7 68 6 4 2338 1 117 Quantibody Rabbit Cytokine Array 1 15 VIII Quantibody Q Analvzer 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 log2. 27 4 82 3 246 9 740 7 2 222 2 6 666 7 20 000 IL 21 0 274 3 823 0 2 469 1 7 407 4 22 222 2 66 666 7 200 000 Leptin 0 137 2 411 5 1 234 6 3 703 7 11 111 1 33 333 3 100 000 MIP 1beta 0 0 5 1 6 4 9 14 8 44 4 133 3 400 MMP 9 0 27 4 82 3 246 9 740 7 2 222 2 6 666 7 20 000 NCAM 1 0 27 4 82 3 246 9 740 7 2 222 2 6 666 7 20 000 TNFalpha 0 5 5 16 5 49 4 148 1 444 4 1 333 3 4 000 Quantibody Rabbit Cytokine Array 1 14 VII Svstem Recoverv 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 cross reactivity with other proteins The spiking recovery rate of the cytokines by the kit in 2x diluted Rabbit serum and cell culture media is listed in the following table Rabbit Cytokine Array I Cross reactivity Test IL IL MIP NCAM TNF CAB DAB lalpha 1beta IL 8 IL 17A IL 21 Leptin 1beta MMP 9 1 alpha IL lalpha 3 888 47 21 57 22 46 46 53 58 30 IL 1beta 63 20 546 1 1 1 1 1 1 1 1 IL 8 25 12 31 440 33 20 49 28 53 99 20 IL 17A 76 26 38 3 424 49 52 87 66 71 62 IL 21 93 127 99 210 2 772 116 147 114 163 106 Leptin 5 1 7 36 3 4 584 16 9 28 1 MIP 1beta 70 75 69 116 102 137 9 934 130 127 89 MMP 9 1 14 7 2 7 3 10 47 919 4 4 NCAM 1 46 29 14 27 37 19 19 16
3. of slides before use mav cause the formation of comet tails B Prepare Cvtokine Standard Dilutions Note There is onlv 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 C Prepare serial dilution of cytokine standards 100ul 100ul 100ul 100Oul 100ul 100ul TS ESOS OO D 8 Add 500ul Sample Diluent 200ul 200u1 200u1 200u1 20041 200u1 100ul Vial Labels Stdi 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 Rabbit Cytokine Array 1 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 100ul Sample Diluent to another tube labeled as CNTRL Do not add standard cytokines or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Note Since the starting concentration of each cytokine is different the serial concentrations from Stdl to St
4. 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 Rabbit Cytokine Array 1 7 IV Protocol A Completelv air drv the glass chip 1 Take out the glass 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 drv at room temperature for another l 2 hours Note Incomplete drving
5. 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 2014 RayBiotech Inc Quantibody Rabbit Cytokine Array 1 21
6. 1 QAM CHE 1 MMP Array QAH MMP 1 Immunoglobin Isotype Array QAH ISO 1 AAM ISO G1 Dried Eye Disease QAH DED 1 Periodontal Disease QAH PDD 1 Sepsis Biomarker QAH SEP 1 Cytokine Number based selection 400 cytokines QAH CAA 9000 360 cytokines QAH CAA 8000 320 cytokines QAH CAA 7000 280 cytokines QAH CAA 6000 240 cytokines QAH CAA 5000 200 cytokines QAH CAA 4000 QAM CAA 4000 160 cytokines QAH CAA 3000 QAM CAA 3000 120 cytokines QAH CAA 2000 QAM CAA 2000 80 cytokines QAH CAA 1000 QAM CAA 1000 60 cytokines QAH ANG 1000 QAM CYT Q2000 40 cytokines QAH INF 3 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CYT 4 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR CYT 3 QAM CHE 1 20 cytokines QAH CYT 1 QAH CYT 2 QAM CYT 1 QAM CYT 2 QAM CYT 3 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 less than 10 cytokines QAH ISO 1 QAH ADI 2 QAP CYT 1 QAM ISO G1 Purpose based selection Custom Arrays Choose from over 1000 cytokine pool Any kind Any number Order slide only or full service in house Desired marker 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 Rabbit Cytokine Array 1 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
7. Quantibody Rabbit Cytokine Array 1 Quantitative measurement of 10 Rabbit cytokines Patent Pending Technology User Manual Version June 2014 Cat QAL CYT 1 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 IL lalpha IL Ibeta IL 8 IL 17A IL 21 Leptin MIP 10 I beta MMP 9 NCAM 1 TNF alpha 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 See Section V For Array Map gt Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody 00000000 00000000 90000000 20000000 t ki 00000000 00000000 m y okine Capture antibody 00000000 00000000 00000000 00000000 00000000 00000000 Glass Slide Support Quantibody Rabbit Cytokine Array 1 1 TABLE OF CONTENTS I OMERVICW i ia ar a Scat te Se Mok fu Introduction vee cto ia HOW IE Works set an SA b A air di p d IL Materials Provided s
8. 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 Rabbit Cytokine Array 1 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 Arravs are not completed covered bv Uneven signal reagent Completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overf
9. 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 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 f
10. d7 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 HO Quantibody Rabbit Cytokine Array 1 9 e Optional for Cell and Tissue Lvsates 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 Completelv remove wash buffer in each wash step Dilute 20x Wash Buff
11. er 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 l 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 Ix 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 Rabbit Cytokine Array 1 10 14 Decant the samples from each well 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 remo
12. ides 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 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 Translati
13. is ista e be To wk sa Additional Materials Required en IIL General Considerations si ia ie A A Preparation of Samples 2sss e neeneenznnza B Handling Glass Chips ccc eeeeee eee ee lied DATION A cate eae Girne neces a IVe Protocol ese aatidatebawciel ace tee gene a era oa A Complete Air Dry the Glass Chip B Prepare Cytokine Standard Dilutions CW N N NANIND DWN WO e C Blocking and Incubation nn D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Dete cil On cncted situ kos ee ines 11 G Data Ahal ysi Sonra a E 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards iva a ess 14 NIL System RECOVELY sisien i jibni ao iz a 15 VIII Quantibody Q Analyzer L 2 eenennenznzzennznn 16 IX Troubleshooting Guide 17 X Select Quantibody Publications 0 ceeeeee eee 18 XI Experimental Record Form 19 XII How to Choose Quantibody Products si f iwa Aarenezzu 20 Quantibody Rabbit Cytokine Array 1 2 I Introduction Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in interactions between different cell types cellular responses to environmental conditions and maintenance of homeostasis In addition cytokines are also involved in most disease processes including
14. known 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 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 Rabbit Cytokine Array 1 4 How It Works Array support YYYYY IC Incubation of Sample FISS With arraved antibodv 1 2 hr Samples 9 Supports Cocktail of Biotin Ab RX KX t KA Incubation with fey oe Biotinylated Ab Labeled E 4 streptavidin l l iq Incubation with vy Cy3 equivalent dye l hr Labeled streptavidin Detection of signals M Data analvsis and graph Quantibody Rabbit Cytokine Array 1 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 dve conjugated Stre
15. ll 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 55 54 57 56 188 178 189 190 Data computation Q Analvzer U Final Result pg ml Quantibody Rabbit Cytokine Array 1 12 V Cytokine Array Map Standard Curves POSI POS2 IL 1 alpha IL 1 beta IL 8 IL 17A IL 21 Leptin MIP 1 beta MMP 9 NCAM 1 TNF alpha QAL CVT 1 Standard Curves 1e 5 1e 4 1e 3 Signal Intensity 1e 2 1e 1 1e 2 1e 1 1e 0 1e 1 1e 2 1e 43 1e 4 1e 5 1e 6 Concentrations pg ml Quantibody Rabbit Cytokine Array 1 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 Limit of Detection LOD Section VIII Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Std1 IL 1alpha 0 13 7 41 2 123 5 370 4 1 111 1 3 333 3 10 000 IL 1beta 0 1 4 4 1 12 3 37 0 111 1 333 3 1 000 IL 8 0 0 3 0 8 2 5 7 4 22 2 66 7 200 IL 17A 0
16. lowing 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 k wash step High E Insufficient wash Increase wash time and use more wash background buffer Dust Work in clean environment Slide is allowed to drv out Don t drv out slides during experiment Quantibody Rabbit Cytokine Array 1 17 Select Quantibodv 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 Neuropept
17. on and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatorv Stimulation Molecular and Cellular Biologv 2008 28 24 7414 7426 11 Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T Ivmphocvtes FEBS Letters 2007 581 26 5087 5093 This reference validates mulitplex ELISA results for several analvtes 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 Rabbit Cytokine Array 1 18 y y y XI Experiment Record Form PMT S Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 EN 2 4 Std5 aj 4 5 Std4 6 Std3 Ls 6 7 Std2 8 Std Ld ID 11 LIL mm 14 15 16 Quantibody Rabbit Cytokine Array 1 19 XII How to Choose Quantibody Products Species based selection Human QAH Mouse QAM Rat QAR Porcine QAP CXT 1 Non Human Primates NHP QAN CYT 1 Canine QAC CYT 1 Feline QAF CYT 1 Equine QAE CYT 1 Bovine QAB CYT 1 Rabbit QAL CYT 1 Function based selection TH1 TH2 TH17 Array QAH TH 1 QAH TH17 QAM TH17 Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 Chemokine Arravs QAH CHE
18. or detection A capture antibody is first bound to the glass surface After incubation with the sample the target cytokine is trapped on the solid surface A second biotin labeled detection antibody is then added which can recognize a different isotope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin labeled Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use array format By arraying multiple cytokine Quantibody Rabbit Cytokine Array 1 3 specific capture antibodies onto a glass support multiplex detection of cvtokines in one experiment is made possible In detail one standard glass slide is spotted with 16 wells of identical cvtokine antibodv arravs Each antibodv 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 un
19. ptavidin 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 l Slide kit 2 Slide kit 1 Quantibody Array Glass Chip 1 2 2 Sample Diluent 1 1 3 20X Wash Buffer I 2 3 4 20X Wash Buffer II 1 1 5 Lyophilized cytokine standard mix 1 1 6 Detection antibody cocktail 1 2 7 Cv3 equivalent dye conjugated Streptavidin 1 2 8 Slide Washer Dryer 1 1 9 Adhesive device sealer 5 10 10 Manual 1 1 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required e Orbital shaker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e 1 5ml Polypropylene microcentrifuge tubes Quantibody Rabbit Cytokine Array 1 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 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
20. ve 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 Ix 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 Ix 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 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 and a low PMT for high signal cytokines Quantibody Rabbit Cytokine Array 1 11 y y y G Data Analvsis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArrav Express ArrayVision or MicroVigene For quantitative data analvsis our Quantibody Q Analyzer software is available It gives visual output as we
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