Human Bone Metabolism Array 2
Contents
1. 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 AS OSOS TS OS GD TN Add 500ul Sample Diluent 2001 200ul 200u1 200ul 20041 20041 100ul 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 Stdl Quantibody Human Bone Metabolism Array 2 8 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes 4 Pipette 100ul 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 Std1 to Std7 for each cytokine are varied whic2. 0 cece cece eee e eee A Preparation of Samples ccc cece eee eens B Handling Glass Chips cece cc aaa C Incubation ccc cece cece cece eee e eee eeeeeees IVe E iOLOCA Lesia iii ii esos a r a EEA A Complete Air Dry the Glass Chip B Prepare Cytokine Standard Dilutions O CO WO N N N ND DW NA W e C Blocking and Incubation 0006 D Incubation with Detection Antibody Cocktail E Incubation with Cy3 Equivalent Dye Streptavidin 10 sy F Fluorescence Detection aaa 11 G Data Analysis kaka ccc eee aaa aaa 12 V Cytokine Array Map amp Standard Curves 13 VI 8 Point Standards kaka kaka 14 VII System Recovery 0 ccc cece cece ence eee aaa 15 VIII Quantibody Q Analyzer J 16 IX Troubleshooting Guide aaa 17 X Select Ouantibody Publications lt 18 XI Experimental Record Form 19 XII How to Choose Ouantibody Products 20 Quantibody Human Bone Metabolism Array 2 I 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
3. QAH ADI 1 QAM INT 2 QAR CYT 1 QAR CYT 2 QAR INF 1 QAN CYT 1 QAP CYT 1 QAH IGF 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 500 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 Ouantibody Human Bone Metabolism Array 2 20 Note Ouantibody is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and 1s 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 2012 RayBiotech Inc Ouantibody Human Bone Metabolism Array 2 21
4. m W E E elelee Quantibody Human Bone Metabolism Array2 19 XII How to Choose Quantibody Products Species based selection Human QAH Mouse QAM Rat QAR CYT 1 QAR CYT 2 QAR CYT 3 QAR INF 1 Porcine QAP CYT 1 Non Human Primates NHP QAN CYT 1 Canine QAC CYT 1 Feline QAF CYT 1 Equine QAE CYT 1 Function based selection TH1 TH2 TH17 Array OAH TH 1 QAH TH17 QAM TH17 Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 OAM INF 1 QAR INF 1 Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 Chemokine Arrays OAH CHE 1 QAM CHE 1 MMP Array OAH MMP 1 Immunoglobin Isotype Array QAH ISO 1 OAM ISO G1 Periodontal Disease Array OAH PDD 1 Bone Metabolism Arrays QAH BMA 1 QAH BMA 2 Cytokine Number based selection 320 cytokines QAH CAA 7000 280 cytokines QAH CAA 6000 240 cytokines QAH CAA 5000 200 cytokines QAH 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 QAH CY T 5 QAH CYT 6 QAH CYT 7 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 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 QAM INT 1 QAH TH17 1 QAM TH17 1 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1
5. slides by the edges only Handle all buffers and slides with latex free gloves Handle glass chip in clean environment Because there 1s 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 1s 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 Human Bone Metabolism Array 2 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 temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry at room temperature for another 1 2 hours Note Incomplete drying of slides before use may cause the formation of
6. Ouantibody Human Bone Metabolism Array 2 Quantitative measurement of 10 human bone metabolism associated cytokines Patent Pending Technology User Manual Version Dec 2012 Cat QAH BMA 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 raybiotech com Email info raybiotech com Cytokine Detected BMP 2 BMP 6 BMP 7 DKK 1 MMP 3 OPG OPN PDGF BB TGFB3 TRANCE One standard glass slide is spotted with 16 wells of 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 See Section V For Array Map 00000000 00000000 00000000 00000000 00000000 00000000 o O 00000000 00000000 00000000 00000000 00000000 00000000 Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody Cytokine _ CC Capture antibody Glass Slide Support Quantibody Human Bone Metabolism Array 2 l TABLE OF CONTENTS l OVELV IE Lis ini ias di ii i as i TAGE OCUICTION sai ns i As How It Works aten tate suchatsenceneiacnaenaadteawisda ius II Materials Provided ccc cece cece eee e eens ee ee aes Additional Materials Reguired 00 II General Considerations
7. er 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 Ouantibody 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 pd 2 3 4 5 6 7 8 9 p lt 2 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Reguired Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Human Bone Metabolism 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 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 B Handling glass chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the
8. etely remove wash buffer in each Hich wash step 18 Insufficient wash Increase wash time and use more wash background buffer Slide is allowed to dry out Don t dry out slides during experiment Ouantibody Human Bone Metabolism Array 2 17 X Select Quantibody 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 HMGBI 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 Sougut 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 Echinacea and anti inflammatory cytokine responses Results of a gene and protein array analysis Pharmacu
9. etical 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 Alzheimer s 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 8 249 251 Quantibody Human Bone Metabolism Array 2 18 XI Experiment Record Form Date File Name Laser Power PMT Well No Sample Name 2 El a E
10. 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 The traditional method for cytokine detection and quantification is through the use of an enzyme linked immunosorbent array ELISA While the traditional method works well for a single protein the overall procedure 1s time consuming and requires a lot of sample Take the advantage of advancement in microarray technology over the last decade 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 Ouantibody 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 for detection A capture antibody is first bound
11. h 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 Quantibody Human Bone Metabolism Array 2 9 shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with H O 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 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 D Incuba
12. iers 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 Ouantibody Human Bone Metabolism Array 2 16 IX Troubleshooting guide Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or Check pipettes and ensure correct improper dilution preparation Weak Signal change sample incubation step to overnight sample sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide 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 Poor standard 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 Compl
13. ism Array 2 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 Human serum SR and 2x diluted Human cell culture media CM is listed in the following table The spiking recovery rate for culture media and serum EMPS 20 000 0 26 834 134 100 000 2 oe 10 000 over over TGFp 10 000 9 6410 64 _ Ouantibody Human Bone Metabolism 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 1s 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 outl
14. lder 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 higher PMT for low signal cytokines and a low PMT for high signal cytokines Ouantibody Human Bone Metabolism Array 2 11 G Data Analysis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArray Express Array Vision or MicroVigene For quantitative data analysis our Ouantibody Q Analyzer software is available It gives visual output as well as digital values Mo
15. okine concentration in the samples will be determined Ouantibody 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 320 human or 160 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 Human Bone Metabolism Array 2 4 How It Works Array support YYYYY B a Samples Incubation of Sample yyy With arrayed antibody 1 2 hr Supports Cocktail of 4 Biotin Ab KX K lt KK Incubation with 1 2 hr Biotinylated Ab Labeled E E E E 4 treptavidi p K K Incubation with 1 hr Aa Cy3 equivalent dye Labeled streptavidin Jose 7 Detection of signals lat Data analysis and graph Quantibody Human Bone Metabolism Array 2 5 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 dye conjugated Streptavidin should be kept at 20 C and all oth
16. re information can be found in section VIII Experiments J Image scan laser scanner J Data extraction 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 GenePix etc ee aeons 55 54 57 56 188 178 189 190 J Data computation Q Analyzer l Final Result pg ml Ouantibody Human Bone Metabolism Array 2 12 V Cytokine Array Map amp Standard Curves BMP 2 BMP 6 QAH BMA 2 Standard Curves BMP 2 BMP 6 BMP 7 DKK 1 MMP 3 OPG OPN PDGF BB TGFb3 TRANCE Signal Intensity Cytokine Concentration pg ml Quantibody Human Bone Metabolism 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 Limit of Detection LOD Section VIII Serial standard concentration pg ml BMP 2 O 14 41 123 370 1 111 3 333 10 000 BMP 6 O 27 82 247 741 2 222 6 667 20 000 BMP 7 O 274 823 2 469 7 407 22 222 66 667 200 000 DKK 1 O 137 412 1 235 3 704 11 111 33 333 100 000 OPG O 27 82 247 741 2 222 6 667 20000 3 8 235 74 222 667 2000 0 0 PDGF BB 0 0 0 Quantibody Human Bone Metabol
17. tion 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 Ouantibody Human Bone Metabolism Array 2 10 14 Decant the samples from each well and wash 5 times with 150 ul of Ix 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 ho
18. 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 Human Bone Metabolism Array 2 3 specific capture antibodies onto a glass support multiplex detection of cytokines in one experiment 1s 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 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 cyt
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