MMI Bibliography II

 

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Laser Microdissection in Cancer Proteomics

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molecular machines industries -bibliography vol 2 no 1 2009 special aacr edition issue on laser microdissection in cancer proteomics

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»lead the way to scientific results« contents in this issue this issue is dedicated to the 100th annual meeting of the american association for cancer research to be held in denver colorado april 18-22 2009 customer reports cancer proteomics by laser microdissection and twodimensional gel electrophoresis comes of age by tadashi kondo md phd proteome bioinformatics project national cancer center tokyo japan measuring targeted therapeutics with targeted metrics combining the precision of laser microdissection with the sensitivity of a nano-immunoassay platform for biomarker discovery by david voehringer phd director applications cell biosciences inc palo alto usa technology product features mmi live cell microdissection ­ the new mmi live cellchamber mmi`s advanced single cell isolation concepts in lasermicrodissection references aacr 2009 american association for cancer research meet the mmi team in denver colorado booth 1020 mmi-bibliography online www.molecular-machines.com/news the lmd-bibliograhy is published by molecular machines industries.com issn 1662-8535 editor dr antje plaschke-schluetter e-mail schluetter@molecular-machines.com editorial board andrea curtis norbert brill andre imhof prof stefan seeger concept layout kathrin plaschke ­ grafik design ­ hamburg the mmi caplift technology is a patented trademark of molecular machines industries www.molecular-machines.com mmi-bibliography 2

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»lead the way together with you« e d i to rial we are happy to introduce to you this second edition of the mmi bibliography this issue illustrates the recent advancements in laser microdissection highlighting the use of mmi products in this field in particular laser microdissection combined with proteomics for the analysis of patient material fosters interesting protocol developments these new protocols allow for the discovery of new biomarkers in cancer and other diseases and are a next step towards personalized medicine tadashi kondo introduces a protocol for the comparative analysis of protein patterns in cancer versus healthy tissue until recently this protocol was used to identify apc-binding protein eb1 as a novel prognostic factor in hepatocellular carcinoma this is a breakthrough workflow for laser microdissection to be used routinely in the clinical environment it emphasizes the reliability robustness and user-friendliness of the mmi systems david voehringer director applications at cellbiosciences inc outlines how laser microdissection in combination with cellbiosciences nano immunoassay reveals detailed information on the phosphorylation status of critical signaling proteins application of a nanoimmunoassay platform to assess changes in egfr-dependent signaling pathways in lung cancer cell lines surgical resections and laser-capture microdissection from patient-derived tumor cells exposed to egfr tyrosine kinase inhibitors mark lloyd andrea w tu jiannong li uyen nguyen soner altiok john koomen lee petruk david w voehringer eric b haura h lee moffitt cancer center tampa fl cell biosciences palo alto ca mmi molecular machines and industries zurich switzerland single cell sorting is a strong focus at mmi and is a prerequisite for various analysis of tumor and rare cells as introduced by tveito et al and pachmann et al array cgh analysis of immunomagnetically isolated breast cancer cells from sentinel lymph node and bone marrow siri tveito leonardo a meza-zepeda Øystein fodstad the norwegian radium hospital oslo norway tracking therapy induced genetic and epigenetic heterogeneity in circulating epithelial tumor cells cetc by single cell genome analysis prediction of therapy success pachmann k camara o hammer u gellner a k rabenstein c runnebaum i b hoeffken department of internal medicine friedrich schiller university jena the whole mmi team hopes that you enjoy this second issue of the mmi bibliography and the aacr meeting 2009 meet us and our customers at our booth 1020 and discover our new developments for non laser based single cell sorting we look forward to seeing you in denver dr antje plaschke-schluetter mmi ag zuerich www.molecular-machines.com mmi-bibliography 3

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»lead the way to cutting edge technologies« c u stom errepo rts cancer proteomics by laser microdissection and two-dimensional gel electrophoresis comes of age laser microdissection is an essential tool in cancer proteomics tumor tissues contain many cell types tumor cells as well as non-tumor cells because of the mixture of cell populations in homogenized tissue proteome data of homogenized tissues does not accurately represent the biological aspects of the cells of interest in addition sample variations will amplify the problem when heterogeneous tissue is recovered in the hospitals and subjected to further analysis thus for accurate and reproducible proteome studies specific and pure cell populations should be recovered using laser microdissection prior to protein extraction two-dimensional gel electrophoresis is one of the most established proteomics tools the idea of using laser microdissection and two-dimensional gel electrophoresis for cancer proteomics was published more than 10 years ago by banks et al [1 however due to the limited sensitivity of spot detection methods such as silver staining laser microdissection was not a practical tool until recently craven et al reported that in the past it took them hours to days to recover enough cells by laser microdissection for proteomic study [2 this throughput was not acceptable in cancer proteomics where as many tissues as possible need to be examined for conclusive results the solution to this situation was an ultra-high sensitive fluorescent dye to quantify the protein samples in 2001 amersham biosciences developed a not yet commercially available fluorescent dye the dye was designed to be 100 times more sensitive at quantifying cystein residues of proteins than conventional silver staining the intended use of this dye was to apply the dye for laser microdissected tissues for cancer proteomics after several trials it was found that the use of the dye dramatically reduced the number of laser microdissected cells needed in 2003 this data was published including the protocol utilizing the dye for laser microdissection and two-dimensional difference gel electrophoresis 2d-dige with dr seike a physician working on lung cancer [3 the dye is now commercially available from ge healthcare biosciences [4 formerly amersham biosciences under the name cydye dige fluor saturation dye for further studies the protocol was optomized for protein annotation using mass spectrometry and database search all detailed protocols are available in a recent publication [5 laser microdissection and 2d-dige are routine tools used in our lab as only 1 mm2 tissue area is needed to generate several thousand protein spots in 2d-dige laser microdissection is now a quick and efficient method to select and collect cells for example dr uemura who joined the esophageal cancer proteomics team [6 applied laser microdissection on 140 samples and completed his analysis in only one month proteome data studied using this approach included biomarker candidates with crucial biological backgrounds for example dr orimo who is a surgeon interested in malignancies of gastrointestinal tracts recently identified apc-binding protein eb1 as a novel prognostic factor in hepatocellular carcinoma [7 identification of www.molecular-machines.com mmi-bibliography 4 tadashi kondo md phd proteome bioinformatics project national cancer center tokyo japan

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»lead the way to advanced proteomics« c u stom errepo rts proteins corresponding to response to treatment metastasis and survival of patients and their clinical application is the research goal of cancer proteomics compared to other proteomic methods the amount of protein required for the experiment in combination with 2d-dige and cydye dige fluor saturation dye is minimal requiring only a few µg on the contrary bai et al reported that they needed 100 µg protein from laser microdissected tissues for liver cancer proteomics using cicat cleavable isotope-coded affinity tags abi framingham ma mass spectrometry-based proteomic technology [8 in conclusion the combination of laser microdissection two-dimensional gel electrophoresis the ultra high sensitive fluorescent dye and mass spectrometry is a highly efficient and recommendable practical approach for tissue cancer proteomics [9 sample material for laser microdissection liver biopsy after h&e-staining references [1 banks r e dunn m j forbes m a stanley a et al the potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis preliminary findings electrophoresis 1999 20 689-700 all tumor tissues from cases with well documented clinico-pathological data [2 craven r a totty n harnden p selby p j banks r e laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis evaluation of tissue preparation and sample limitations am j pathol 2002 160 815-822 [3 kondo t seike m mori y fujii k et al application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool proteomics 2003 3 1758-1766 [4 shaw j rowlinson r nickson j stone t et al evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes proteomics 2003 3 1181-1195 hematoxylin stained control picture after laser microdissection collected material on the cap surface [5 kondo t hirohashi s application of highly sensitive fluorescent dyes cydye dige fluor saturation dyes to laser microdissection and two-dimensional difference gel electrophoresis 2d-dige for cancer proteomics nat protoc 2006 1 2940-2956 [6 uemura n nakanishi y kato h saito s et al transglutaminase 3 as a prognostic biomarker in esophageal cancer revealed by proteomics int j cancer 2008 [7 orimo t ojima h hiraoka n saito s et al proteomic profiling reveals the prognostic value of adenomatous polyposis coli-end-binding protein 1 in hepatocellular carcinoma hepatology 2008 48 1851-1863 [8 bai d s dai z zhou j liu y k et al capn4 overexpression underlies tumor invasion and metastasis after liver transplantation for hepatocellular carcinoma hepatology 2009 49 460-470 [9 kondo t tissue proteomics for cancer biomarker development laser microdissection and 2d-dige bmb rep 2008 41 626-634 www.molecular-machines.com mmi-bibliography 5

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»lead the way to cutting edge technologies« c u stom errepo rts measuring targeted therapeutics with targeted metrics combining the precision of laser microdissection with the sensitivity of a nano-immunoassay platform for biomarker discovery exciting progress and tremendous efforts are being dedicated to the development of anti-cancer therapies that target the molecular sequela that underlie carcinogenesis tyrosine kinase inhibitors tkis such as imatinib gleevec have been shown to have dramatic effects in individuals suffering from chronic myelogenous leukemia cll gastrointestinal stromal tumors gist and are being evaluated in a number of tumor types by targeting inappropriate tyrosine kinase signaling these compounds trigger apoptosis resulting in decrease in tumor burden since this drug development approach is predicated on the idea of targeting the molecular biology at the site of disease novel methods are required to evaluate the effectiveness of new therapies cell-based assays are often used during early phases of development however here there is little heterogeneity and usually sufficient material for multi-faceted evaluation ultimately however these compounds are evaluated in the clinical setting where material is often heterogeneous and limited therefore tools and/or methods that reduce heterogeneity and enable as many endpoints as possible with this precious material are desired laser capture microdissection lcm has proven to be an excellent tool for isolating specific populations of cells from heterogeneous tissue structure specifically different regions of a tumor can be isolated from tissue sections hypoxic versus non-hypoxic for instance as well as micro-environment structure normal tissue vasculature etc technologies for lcm vary and have tradeoffs for different applications for example collection methods of nucleic acid for amplification have been developed for most lcm tools however broad success of protein extraction for analysis is limited 4x david voehringer phd director applications cell biosciences inc palo alto usa the hetrerogeneity of primary tumour tissue only a percentage of material is nsclc tumorareal inflammatory infiltrate 40x the major factor influencing lcm selection is the lack of methods for amplifying protein signal therefore methods for material acquisition must take into account efficiency of protein extraction and quality of extraction these criteria limit the lcm technologies that can be applied ideally to prevent protein degradation the method is rapid and has little impact on the protein i.e protein dephosphorylation or other non-biological change we have developed a protocol using the mmi cellcut plus microdissection system molecular machines industries that allows for rapid isolation of tissue sections as small as 500 cells this provides sufficient material for protein evaluation in a state that maintains the phosphorylation of critical signaling proteins [2 importantly this method is rapidly performed on oct embedded biopsies under controlled conditions yet still allows downstream extraction of proteins in order to measure the proteins of interest a method for analyzing small samples of material is necessary we employed cell biosciences nano-immunoassay platform because of its ability to detect femtogram levels of protein and its unique ability to reproducibly quantitate phosphorylation state of a protein due to changes in protein www.molecular-machines.com mmi-bibliography 6 a 0.8 mm core biopsy sample from phase ii clinical trial being run at moffittcancer center for further details see also lloyd et al aacr 2009

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»lead the way to microproteomics« c u stom errepo rts charge [1 briefly 200 nl or less of sample is drawn into a 5 cm x 100 micron inner-diameter capillary 400 nl total volume along with electrophoresis reagents an electric field is applied and isoelectric focusing ief occurs as proteins concentrate at their net neutral charge or pi proteins are immobilized to the wall of the capillary through a photoactivation capture chemistry step lastly a traditional immunoassay is performed with a primary antibody to the protein of interest anti-total erk in the example provided and hrp-conjugated reporter secondary antibody due to the fact that addition of a phosphate to a protein alters a proteins charge the resulting data is a distribution of non-phosphorylated as well as phosphorylated isoforms area-under-the-peak measurements are extracted and total as well as percent phospho-isoform distribution of the specific protein are quantitated in a collaborative effort with dr eric haura and colleagues at moffitt cancer center protein activity was measured in non-small cell lung cancer patients from both tumor core biopsies collected by lcm as well as subsequent surgical resection [2 we feel that this a promising technique that is minimally invasive and could provide a window into tumor response to targeted therapy biopsies could be taken prior to the initiation and throughout the course of therapy and be used as either a prospective technique of drug effectiveness as well as eventually a method to tailor therapy to an individual patients response [3 mmi cellcut plus laser capture microdissection of patient ncslc biopsies punch biopsy from patient with nsclc a primary tissue section prior to lcm b same section after removal of tumor-enriched material c adjacent serial section stained with h&e d overlay of stained and lcm serial sections to verify section purity for tumor cells a b c read out of a cellbiosciences nanoimmunoassay activity of erk isoforms varies in different patients references [1 o neill r a bhamidipati a bi x deb-basu d cahill l ferrante j gentalen e glazer m gossett j hacker k et al 2006 isoelectric focusing technology quantifies protein signaling in 25 cells proc natl acad sci.103:16153­16158 [2 haura e b lloyd m tu a w li j sommers k e altiok s nguyen u petruk l koomen j voehringer d w application of a nano-immunoassay platform to evaluate erk signaling in human lung cancer specimens using less than 500 cells in preparation d [3 koomen j m haura e b bepler g sutphen r remily-wood e r benson k hussein m hazlehurst l a yeatman t j hildreth l t sellers t a jacobsen p b fenstermacher d a dalton w s proteomic contributions to personalized cancer care mol cell proteomics 2008 7:1780-1794 www.molecular-machines.com mmi-bibliography 7

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»lead the way to live cell microdissection« techno lo g y productfe at ures mmi live cell microdissection ­ the new mmi live cellchamber introduction the isolation and enrichment of individual live cells for culture and differentiation experiments or for their proteomic and genomic analysis is of increasing interest in stem cell research cancer research and tissue engineering while mechanical separation techniques are cumbersome time consuming and bear the risks of contamination or mechanical stress faster laser microdissection based methods are currently under development the mmi cellcut plus in combination with the newly developed mmi live cellchamber enables contamination-free isolation of cells in living culture using the mmi live cellchamber seed cells and cultivate to the desired cell density visualize your cells of interest e.g phase contrast immunolabeling etc transfer the membrane ring with cells into the mmi microdissection chamber components of the new mmi live cellchamber · a membrane ring for the initial cultivation of cells · a cell culture dish to house the membrane ring with seeded cells · the microdissection chamber uv ­ permeable and filled with adhesive for laser microdissection of single cells or group of cells note all components are sterilized and ready to use the workflow adherent cells are grown in a special metal ring on a membrane inside a petri dish once a desired cell density is reached the membrane ring is transferred to the adhesive area of the microdissection chamber the cell or cells of interest are selected and cut using laser microdissection in addition unwanted cells can be destroyed or ablated by using individual laser shots the cell chamber is then removed and only the cells of interest remain in the microdissection chamber repeated selection of wanted cells this process can be repeated using the same metal ring in a new microdissection chamber to select and separate more than one cell type once isolation is complete sufficient medium is added to the microdissection chamber and the cells can be re-cultivated selection of cells via lmd your advantage the isolation of single cells is a unique and highly useful tool in wide range of research and clinical applications including individual cell analysis clonal expansion transfection experiments and stem cell research enzyme treatments i.e trypsin other potentially harmful selective reagents as well as time consuming repetitive enrichments can be avoided the benefit is an increase in reliability effectiveness and accuracy of your research order this product and report back to us every feedback will be rewarded isolate cells by removing the membrane ring with unwanted cells reculture the cells of interest and if desired the depleted fraction repeat if desired order nr 50301 www.molecular-machines.com mmi-bibliography 8

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»lead the way to integrated work flows« techno lo g y productfe at ures mmi s advanced single cell isolation concepts in laser microdissection single cell isolation in combination with laser microdissection is of growing interest mainly the analysis of tissue invading cells like immune cells vascular cells invading metastatic cells and tissue or cancer stem cells are of interest see fevr et al 2007 likewise single cells from blood or bone marrow smears or any other aspirate can be microdissected prior to downstream analysis in cytogenetics and biomedical research the main advantage of laser microdissection over other methods like facs and beadbased sorting lies in the unsurpassed high precision and visual control of the selection and isolation process in order to make single cell isolation reproducible and reliable mmi established advanced concepts for single cell dissection and collection settings for ptp-collection generate an overview screen of a sample a bloodsmear is displayes here ptp see what you get and get what you see predefined target positioning is a new patented product feature that allows for the precise predefined contamination free and visually controlled collection of individual cells the workflow the experiment to the left illustrates the collection of individual granulocytes from a blood smear first an overview screen of the whole sample is produced second visual inspection and selection of the target cells is performed hint this step can be fully automated using the mmi cellexplorer software the uv-cut software is than adjusted to the ptp mode and cells 1 to 100 are dissected and collected autodocumentation ensures accurate in process control for the evaluation of the experiment at a later time dr hussein et al have successfully established single cell sorting concepts for the molecular analysis of single cells in hematological diseases of myeloid origin cml p vera and others thus mmi`s ptp function provides absolute reliability and visual control of the cell sorting process adjust detailed settings for defined collection of target cells references dissect and control collected cells on the adhesive cap fevr t robine s louvard d and huelsken j 2007 wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells mol cell biol 27 7551-7559 hussein k bock o theophile k seegers a arps h basten o grips k h franz-werner j büsche g kreipe h 2008 chronic myeloproliferative diseases with concurrent bcrabl junction and jak2v617f mutation leukemia 2008 may 225 1059-62 hussein k dralle w theophile k kreipe h bock o 2008 megakaryocytic expression of mirna 10a 17-5p 20a and 126 in philadelphia chromosome-negative myeloproliferative neoplasm ann hematol 2009 apr 884 325-32 epub 2008 sep 5 hussein k von neuhoff n büsche g buhr t kreipe h bock o opposite expression pattern of src kinase lyn in acute and chronic haematological malignancies ann hematol 2009 mar 17 www.molecular-machines.com mmi-bibliography 9

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»lead the way to an easy operation« references ashida s furihata m katagiri t tamura k anazawa y yoshioka h miki t fujioka t shuin t nakamura y and nakagawa h 2006 ashida s nakagawa h katagiri t furihata m iiizumi m anazawa y tsunoda t takata r kasahara k miki t fujioka t shuin t and nakamura y 2004 bohm m wieland i schutze k and rubben h 1997 buckanovich r j sasaroli d o`brien-jenkins a botbyl j conejo-garcia j r benencia f liotta l a gimotty p a and coukos g 2006 buckanovich r j sasaroli d o`brien-jenkins a botbyl j hammond r katsaros d sandaltzopoulos r liotta l a gimotty p a and coukos g 2007 cowherd s m espina v a petricoin e f iii and liotta l a 2004 dahl e kristiansen g gottlob k klaman i ebner e hinzmann b hermann k pilarsky c durst m klinkhammerschalke m blaszyk h knuechel r hartmann a rosenthal a and wild p.j 2006 espina v dettloff k a cowherd s petricoin e f iii and liotta l a 2004 espina v heiby m pierobon m and liotta l a 2007 espina v wulfkuhle j d calvert v s vanmeter a zhou w coukos g geho d h petricoin e f iii and liotta l a 2006 fevr t robine s louvard d and huelsken j 2007 expression of novel molecules mical2-pv mical2 prostate cancer variants increases with high clin cancer res 12 2767-2773 gleason score and prostate cancer progression molecular features of the transition from prostatic intraepithelial neoplasia pin to prostate cancer genome-wide gene-expression profiles of prostate cancers and pins cancer res 64 5963-5972 microbeam moment non-contact laser microdissection of membrane-mounted native tissue am j pathol 151 63-67 use of immuno-lcm to identify the in situ expression profile of cellular constituents of the tumor cancer biol ther 5 635-642 microenvironment tumor vascular proteins as biomarkers in ovarian cancer j clin oncol 25 852-861 proteomic analysis of human breast cancer tissue with laser-capture microdissection and reverseclin breast cancer 5 385-392 phase protein microarrays molecular profiling of laser-microdissected matched tumor and normal breast tissue identifies karyopherin alpha2 as a potential novel prognostic marker in breast cancer clin cancer res 12 3950-3960 use of proteomic analysis to monitor responses to biological therapies expert opin biol ther 4 83-93 laser capture microdissection technology laser-capture microdissection expert rev mol diagn 7 647-657 nat protoc 1 586-603 wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells mol cell biol 27 7551-7559 gjerdrum l m abrahamsen h n villegas b sorensen b s schmidt h and hamilton-dutoit s j 2004 gjerdrum l m and hamilton-dutoit s 2005a the influence of immunohistochemistry on mrna recovery from microdissected frozen and formadiagn mol pathol 13 224-233 lin-fixed paraffin-embedded sections laser-assisted microdissection of membrane-mounted sections following immunohistochemistry methods mol biol 293 139-149 and in situ hybridization laser-assisted microdissection of membrane-mounted tissue sections methods mol biol 293 127-138 gjerdrum l.m and hamilton-dutoit s 2005b gjerdrum l m lielpetere i rasmussen l m bendix k and hamilton-dutoit s 2001 hatakeyama h kondo t fujii k nakanishi y kato h fukuda s and hirohashi s 2006 kasper g vogel a klaman i grone j petersen i weber b castanos-velez e staub e and mennerich d 2005a kasper g weiser a a rump a sparbier k dahl e hartmann a wild p schwidetzky u castanos-velez e and lehmann k 2005b kondo t and hirohashi s 2006 laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis identification of cell populations using immunohistochemistry and in situ j mol diagn 3 105-110 hybridization protein clusters associated with carcinogenesis histological differentiation and nodal metastasis proteomics 6 6300-6316 in esophageal cancer the human laptm4b transcript is upregulated in various types of solid tumours and seems to play cancer lett 224 93-103 a dual functional role during tumour progression expression levels of the putative zinc transporter liv-1 are associated with a better outcome of int j cancer 117 961-973 breast cancer patients application of highly sensitive fluorescent dyes cydye dige fluor saturation dyes to laser microdissection and two-dimensional difference gel electrophoresis 2d-dige for cancer proteomics nat protoc 1 2940-2956 kondo t seike m mori y fujii k yamada t and hirohashi s 2003 kube d m savci-heijink c d lamblin a f kosari f vasmatzis g cheville j c connelly d p and klee g g 2007 kumar-sinha c shah r b laxman b tomlins s a harwood j schmitz w conzelmann e sanda m.g wei j t rubin m a and chinnaiyan a m 2004 application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional proteomics 3 1758-1766 gel electrophoresis as a cancer proteomic study tool optimization of laser capture microdissection and rna amplification for gene expression profiling bmc mol biol 8 25 of prostate cancer elevated alpha-methylacyl-coa racemase enzymatic activity in prostate cancer am j pathol 164 787-793 www.molecular-machines.com mmi-bibliography 10

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»lead the way to microscopic manipulation« references moreno-bueno g hardisson d sanchez c sarrio d cassia r garcia-rostan g prat j guo m herman j g matias-guiu x esteller m and palacios j 2002 obama k ura k li m katagiri t tsunoda t nomura a satoh s nakamura y and furukawa y 2005 orimo t ojima o hiraoka n saito s kosuge t kakisaka t yokoo h nakanshi k karniyama t todo s hirohashi s kondo t 2008 pugh t j bebb g barclay l sutcliffe m fee j salski c o`connor r ho c murray n melosky b english j vielkind j horsman d laskin j j and marra m a 2007 sanchez-aguilera a delgado j camacho f i sanchez-beato m sanchez l montalban c fresno m f martin c piris m a and garcia j f 2004 scarpino s cancellario d f di n a pasquini a marzullo a and ruco l p 2004a scarpino s di n a melotti f talerico c cancrini a and ruco l 2007a scarpino s di n a rapazzotti-onelli m pilozzi e and ruco l 2004b scarpino s di n a stoppacciaro a antonelli m pilozzi e chiarle r palestro g marino m facciolo f rendina e a webster k e kinkel s a scott h s and ruco l 2007b scarpino s di n a taraboletti g cancrini a and ruco l p 2005 schipper h papp t johnen g pemsel h bastrop r muller k m wiethege t jaworska m krismann m schiffmann d and rahman q 2003 takata r katagiri t kanehira m tsunoda t shuin t miki t namiki m kohri k matsushita y fujioka t and nakamura y 2005 tamura k furihata m tsunoda t ashida s takata r obara w yoshioka h daigo y nasu y kumon h konaka h namiki m tozawa k kohri k tanji n yokoyama m shimazui t akaza h mizutani y miki t fujioka t shuin t nakamura y and nakagawa h 2007 tomlins s a mehra r rhodes d r cao x wang l dhanasekaran s m kalyana-sundaram s wei j t rubin m va pienta k j shah r.vb and chinnaiyan a m 2007 tomlins s a mehra r rhodes d r shah r b rubin m a bruening e makarov v and chinnaiyan a m 2006 tveito s maelandsmo g m hoifodt h k rasmussen h and fodstad o 2007 wang l and zhu h 2006 abnormalities of the apc/beta-catenin pathway in endometrial cancer oncogene 21 7981-7990 genome-wide analysis of gene expression in human intrahepatic cholangiocarcinoma hepatology 41 1339-1348 proteomic profiling reveals the prognostic value of adenomatous polyposis coli-end-binding protein 1 in hepatocellular carcinoma journal of hepatology in press correlations of egfr mutations and increases in egfr and her2 copy number to gefitinib response cancer 7 128 in a retrospective analysis of lung cancer patients bmc silencing of the p18ink4c gene by promoter hypermethylation in reed-sternberg cells in hodgkin blood 103 2351-2357 lymphomas increased expression of met protein is associated with up-regulation of hypoxia inducible factor-1 j pathol 202 352-358 hif-1 in tumour cells in papillary carcinoma of the thyroid papillary carcinoma of the thyroid low expression of ncam cd56 is associated with downregulaj pathol 212 411-419 tion of vegf-d production by tumour cells papillary carcinoma of the thyroid methylation is not involved in the regulation of met br j cancer 91 703-706 expression expression of autoimmune regulator gene aire and t regulatory cells in human thymomas clin exp immunol 149 504-512 hepatocyte growth factor hgf downregulates thrombospondin 1 tsp-1 expression in thyroid papillary carcinoma cells j pathol 205 50-56 mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human int j oncol 22 1009-1017 malignant mesotheliomas predicting response to methotrexate vinblastine doxorubicin and cisplatin neoadjuvant chemotherapy for bladder cancers through genome-wide gene expression profiling clin cancer res 11 2625-2636 molecular features of hormone-refractory prostate cancer cells by genome-wide gene expression cancer res 67 5117-5125 profiles integrative molecular concept modeling of prostate cancer progression nat genet 39 41-51 whole transcriptome amplification for gene expression profiling and development of molecular neoplasia 8 153-162 archives specific isolation of disseminated cancer cells a new method permitting sensitive detection of clin exp metastasis 24 317-327 target molecules of diagnostic and therapeutic value clonal analysis of palmar fibromatosis a study whether palmar fibromatosis is a real tumor j transl med 4 21 yamabuki t daigo y kato t hayama s tsunoda t miyamoto m ito t fujita m hosokawa m kondo s and nakamura y 2006 zhang l yang n conejo-garcia j r katsaros d mohamed-hadley a fracchioli s schlienger k toll a levine b rubin s c and coukos g 2003a zhang l yang n park j.w katsaros d fracchioli s cao g o`brien-jenkins a randall t c rubin s c and coukos g 2003b genome-wide gene expression profile analysis of esophageal squamous cell carcinomas int j oncol 28 1375-1384 expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma clin cancer res 9 264-272 tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature supporting angiogenesis in ovarian cancer cancer res 63 3403-3412 to be continued www.molecular-machines.com mmi-bibliography 11

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s pecific at ions mmi cellcut plus item system components samples microscopic systems picosecond uva solid-state laser mmi caplift technology nosepiece uis2 objectives digital camera with ultra high sensitivity mmi uv-cut software for all application-relevant samples cryo or paraffin-preserved tissues single cells cell compartiments cytospins chromosomes etc olympus ix71 or ix81 or nikon eclipse computer-controlled wavelength 355 nm pulse duration 500 psec pulse energy/average energy 1 µjoule/appr 4 mw repetition rate 5 khz sw-controlled covering full slides unique contamination-free sandwich technology 6-position nosepiece na and wd specified to be selected according to application requirements excellent uv/ir transmission digital colour digital monochrome 1,392 x 1,040 pixels compact housing and firewire connection laser energy and focus control full slide and petri dish control inspection mode with positive target identification saving multi user profiles multigroup function across entire sample/slides autodocumentation for sample images and parameters pc and monitor motorised stage motorised nosepiece z-focus for ix81 condenser/contrast methods fluorescence options penscreen system operation mmi cellexplorer image analysis software mmi multicap motorised mmi multislide motorised possible cellcut system upgrade mmi cellmanipulator optical tweezers new mmi cellector ultra precise contact-free manipulation of microscopic particles with up to 10 independent beams based on a high-quality yag-type infrared laser a fully automated single cell sorting device sensitive 21 pen screen monitor for user-friendly system operationand to allow direct target identification with a special pen identifies and cuts out automatically defined targets based on user settings allows automatic collection of targets in up to 8 different isolationcaps for microdissection of up to 3 slide assemblies 10 nm step size and 1 µm reproducibility independent of movement direction fine/coarse movement with 3 mm/sec max speed bf phase and rc contrast and dic application-oriented 6-cube turret dual-level laser coupling specifications will be continuously updated according to market development windows xp 19 lcd monitor computer-controlled for high-precision movement/cutting travelling range 120 x 100 mm step width 0.075 µm repositioning accuracy 1 µm specification the manufacturer reserves the right to make technical changes without prior notice mmi ag flughofstrasse 37 8152 glattbrugg switzerland phone +41 44 809 10 10 fax +41 44 809 10 11 mail info@molecular-machines.com www.molecular-machines.com mmi gmbh breslauer strasse 2 85386 eching germany phone +49 89 319 048 40 fax +49 89 319 048 59 mail info@molecular-machines.com www.molecular-machines.com mmi inc p.o box 348 haslett mi 48840 usa phone +1 603 629 95 36 fax +1 321 978 0304 april 2009 mail sales_us@molecular-machines.com www.molecular-machines.com

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