User Manual - RayBiotech, Inc.
Contents
1. paste process the cytokine expression levels are determined per sample 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 numbers of samples e Two Positive Controls The program utilizes the two positive controls in each array for normalization e User Intervention The program allows for user manual handling of outliers and other analytical data e Analyze Multiple Slide The data for multiple slides can be inputted for easy slide to slide comparison XI Troubleshooting Guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent Check pipettes and ensure correct volumes or improper dilution preparation E rr Increase incubation time or change sample hort in Weak Signal Sonne bana mine incubation step to overnight Too low protein Lessen dilution or do not dilute sample concentration in sample Concentrate sample if necessary inbraparsiorida cri Store kit as suggested temperature Don t BISP 9 freeze thaw the slide Bubble formed during Decrease amount of rocking shaking during incubations check for bubble formation and incubation remove bubbles E Arrays are not Uneven signal completed covered by Completely cover arrays with solution for all required steps reagent Paaca2. 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 VIII Protocol A Completely Air Dry The Glass Slide 1 Take out the glass slide 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 for another 1 2 hours Incomplete drying of slides before use may cause the formation of comet tails thin directional smearing of antibody spots B Blocking amp Incubation 2 Add 100 ul Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides 3 Decant buffer from each well Add 100 ul of sample to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals This step may be done overnight at 4 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 especially if less than 70 ul of sample or reagent is used 4 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of 1X Wash Buffer at room temper
3. 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 10 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step E Fluorescence Detection 11 12 13 14 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 Place the slide in the Slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x Wash Buffer 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 and gently shake at room temperature for 5 minutes Remove water droplets completely by gently applying suction with a pipette to remove water droplets Do not touch the array only the sides You may also dry the glass slide by a compressed N2 stream Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength green channel such as Axon GenePix 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 s
4. G Series Human Bone Metabolism Array 1 Semi quantitative measurement of 31 human bone metabolism associated cytokines Catalog GSH BMA 1 User Manual Last revised May 2015 Caution Extraordinarily useful information enclosed Ke RayBiotech The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com 1 mm o D O O O S D S WD Page D Additional Materials Required TETT VI VII General Considerations A Sample Preparation B Handling Glass Slides C Incubation Protocol A Completely Air Dry The Glass Slide B Blocking amp Incubation C Incubation with Biotinylated Antibody Cocktail amp Wash D Incubation with Cy3 Equivalent Dye Streptavidin amp Wash E Fluorescence Detection F Data Analysis EEE ll Select Publications H E X X gt N NN q q qq gt Q Q m gt Q N oO Please read the entire manual carefully before starting your experiment I Overview Activin A aFGF FGF 1 Amphiregulin DFGF BMP 4 BMP 9 E Selectin ICAM 1 CD54 IGF 1 IL 1 alpha IL 1 F1 IL 1 beta IL 1 F2 IL 6 IL 8 CXCL8 IL 11 IL 17A MCP 1 Cytokines CCL2 M CSF MIP 1 alpha CCL3 MMP 2 MMP 9 MMP Detected 13 Osteoactivin GPNMB P Cadherin RANK 31 TNFRSF11A SDF 1 alp
5. a and anti inflammatory cytokine responses Results of a gene and protein array analysis Pharmacuetical Biology 2009 47 6 500 508 7 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 8 Du et al P2 380 Identification and characterization of human autoantibodies that may be used for the treatment of prion diseases Alzheimers and Dementia 2009 4 4 T484 T484 9 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 2497251 Note The citations listed above are for the Quantibody product l
6. ature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer with H20 e Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1X Wash Buffer cover the whole glass slide and frame with Wash Buffer and wash at room temperature with gentle shaking for 20 min e Decant the 1x Wash Buffer 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 H20 Incomplete removal of the wash buffer in each wash step may cause dark spots the background signals higher than the spots C Incubation with Biotinylated Antibody Cocktail amp Wash 5 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 6 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 7 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer 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 D Incubation with Cy3 Equivalent Dye Streptavidin amp Wash 8 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 9 Add 80
7. es chemokines growth 3 factors proteases soluble receptors and other proteins from any biological fluid Like a traditional sandwich based ELISA this array uses a matched pair of cytokine specific antibodies for detection After incubation with the sample the target cytokines are captured by the antibodies printed on the solid surface A second biotin labeled detection antibody is then added which recognizes a different epitope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin conjugated Cy3 equivalent dye Like the Quantibody arrays G Series utilizes a highly sensitive and stable fluorescent readout which can be detected by most laser fluorescent scanner systems After capturing the spot densities with a laser scanner normalization of the raw data can be easily calculated by the researcher or by a quick copy paste into our excel based Analysis Tool software This array as well as all catalog numbers beginning with GS differ from the classic G Series Arrays in a few important ways First each capture antibody is printed in quadruplicate instead of duplicate delivering higher precision Secondly this array features the same antibody panels used in our Quantibody Arrays allowing a seamless transition to our quantitative multiplex assay platform Lastly all 16 wells are spotted as sub arrays delivering easy handling of 16 samples simultaneously while consumi
8. ha CXCL12 alpha Sonic Hedgehog N Terminal Shh N TGF beta 1 TGF beta 2 TNF alpha VCAM 1 CD106 VE Cadherin CDH5 See Section IX for Array Map One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Detection Fluorescence Go to www RayBiotech com Scanners for a Method list of compatible laser scanners Sample Volume 50 100 ul per array Reproducibility CV lt 20 Il Introduction Bone is a metabolically active tissue that undergoes continuous remodeling by two counter acting processes namely bone formation osteoblasts and bone resorption osteoclasts Under normal conditions bone resorption and formation are tightly regulated by various hormones e g PTH vitamin D steroids and calcitonin and local mediators e g cytokines and growth factors Bone resorption requires the presence of RANKL and M CSF and is inhibited by OPG Bone formation is induced by many growth factors in particular the BMPs FGF PDGF and TGFb and is regulated by M CSF ALP osteocalcin osteopontin and osteonectin An imbalance in the regulation of bone resorption and bone formation results in many metabolic bone diseases such as osteoporosis osteoarthritis rheumatoid arthritis and bone metastases RayBio G Series Arrays are glass slide based antibody arrays which allow researchers to conduct rapid accurate expression profiling of hundreds of cytokin
9. ignal cytokines F Data Analysis 15 Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software GenePix ScanArray Express ArrayVision MicroVigene etc GAL files can be found here www RayBiotech com Gal Files html Need help analyzing all that data Copy and paste your data into the Q Analyzer Tool specific for this array catalog number GSH BMA 1 SW More information can be found in Section X Experiment Image Scan A list of compatible laser scanners can be found here RayBiotech com Scanners Data Extraction GenePix etc Analyze Results Analysis Tool sold separately see Section X IX Array Map LEPE La te ae FE SE ae aE Se EZ PA POST POS2 ActivinA B aFGF FGF 1 gt Amphireguin bFGF c BMP4 BMPS gt E Seledtin gt D ICAM1 CD54 IGF LM alpha E beta LG ILB CXCLB ES MM NATA MCP 1 CCL2 e mes MIP alpha COL3 _ MMe2 H MMP9 MMP13 Osteoactivin Pacha RANK SF alpha X Array Data Analysis Tool The RayBio Analysis Tools are array specific Excel based program that perform sophisticated data analysis on the raw numerical data extracted from the array scan see below for description The Analysis Tool specific for this array is catalog number GSH BMA 1 SW Key features e Simplicity Easy to operate and requires no professional training With a simple copy and
10. ine which is the same as the GS Series but include protein standards for quantitation XIII Experiment Record Form Date File Name Laser Power PMTL ooo Weli No o mm man EM nm KE pele de Leslee lls U E E 2 XIV How to Choose a GS Series Array Species based selection Human GSH Mouse GSM Rat GSR Bovine GSB Canine GSC Equine GSE Feline GSF Ovine GSO Primates GSN Porcine GSP Rabbit GSL Function based selection Adhesion Molecule Angiogenesis Arrays Bone Metabolism Chemokine Arrays Arrays Arrays El Biomarker Custom Arrays Cytokine Arrays Growth Factor Arrays aP neno IL 1 Family Arrays mne nesponce Inflammation Arrays Arrays Arrays Interleukin Arrays Isotyping Arrays MMP Arrays Obesity Arrays Ophthalmic Arrays Mo Disgagg Receptor Arrays Th1 Th2 Th17 Arrays Cytokine Number based selection Arrays are available in the GS Series amp Quantibody platform to detect 660 human 200 mouse or 67 rat proteins GLP Compliant testing services are also available This product is for research use only 2015 RayBiotech Inc
11. ng low sample volumes 10 100 ul per array ll How It Works Sample te e Y Y Y e k Pt Antibody array support a Y Y Y glass slide Incubation of sample T of t a 1 2 hours Incubation with biotinylated E antibody cocktail 7 Y Y 1 2 hours ti Incubation with Cy3 equivalent dye labeled streptavidin 1 hour Scan and perform data extraction amp analysis IV Materials Provided Catalog Component Name 1 Slide Box pons GSH BMA 1S Brad Bone Metabolism Array 1 Glass AA WB1 30ML 20X Wash Buffer 2 x 30 ml 3 x 30 ml AA WB2 30ML 20X Wash Buffer II Human Bone Metabolism Array 1 Sree Rs Biotinylated Antibody Cocktail eo Ereni Cy3 equivalent dye conjugated QA SWD Slide Washer Dryer 1 x 30 ml Tube 4 slide kits are comprised of 2 separate 2 slide kits V Storage Upon receipt all components should be stored at 20 C The kit will retain activity for up to 6 months Once thawed the glass slide antibody cocktail and dye conjugated Streptavidin should be kept at 20 C All other components may be stored at 4 C The entire kit should be used within 6 months of purchase VI Additional Materials Required Benchtop rocker or orbital rocker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5 ml Polypropylene microcentrifuge tubes VII General Considerations A Preparation of Samples e Use serum free conditioned media if possible e f serum contai
12. ning conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contains cytokines e We recommend the following parameters for your samples 50 to 100 ul of original or diluted serum plasma cell culture media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates If you experience high background or if the fluorescent signal intensities exceed the detection range further dilution of your sample is recommended B Handling Glass Slides e Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only e Handle all buffers and slides with powder free gloves Handle glass slide s in clean environment The GS Series slides do not have bar codes To help distinguish one slide from another transcribe the slide serial number from the slide bag to the back of the slide with a fine point permanent marker Please write the number on the very bottom edge of the slide taking care to avoid writing on the array well areas C Incubation e Completely cover array area with sample or buffer during incubation e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rocking or rotation e 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 e Several incubation
13. nkevaborniion Cover the incubation chamber with 9 p adhesive film during incubation Overexposure Lower the PMT or sigmal gain akspis Completely remove wash buffer in each wash step High Increase wash time and use more wash Insufficient wash background buffer Dust Work in clean environment rag slove og Don t dry out slides during experiment XII Select Publications 1 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 2 Willingham SB et al NLRP3 NALP3 Cryopyrin facilitates in vivo caspase 1 activation necrosis and HMGB1 release via inflammasome dependent and independent pathways J Immunol 2009 183 3 2008 15 3 El Karim et al Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 4 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 5 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 6 Altamirano Dimas et al Echinace
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