Cancer Research Biotec

Discover and advance

Cancer research

Dissociate tumor tissue

Isolate cancer stem cells

Enrich and detectcirculating tumor cells

Investigate endothelial and progenitor cells

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MACS® Technology by Miltenyi BiotecProviding a firm foundation for reliable results

Propel your cancer research with MACS® Technology MACS® Technology, the recognized standard in cell separation, has supported oncology research and immunobiology for over 20 years.

Benefit from MACS Technology• Easilyisolaterarecellpopulations

• Optimalrecoveryandexcellentpurity

• Fast,convenient,andreliable

• Gentletocells

• Automatedcellseparationwiththe autoMACS® Pro Separator

• Compatiblewithflowcytometry

• Cellseparationscaneasilybescaled-up

• Bridgesbasicresearchandclinicalapplications

About MACS MicroBeadsMACSMicroBeadsaresuperparamagneticparticlesofapproximately50nanometersindiameter.Theyarecomposedofabiodegradablematrix,anditisthereforenotnecessarytoremove them from cells after the separation process.

• Colloidal,foreasyhandling,andshortincubationtimes

• Small(50nm),non-toxic,biodegradable

• Detachmentisnotrequiredfordownstreamexperiments

• Conjugatedtohighlyspecificmonoclonalantibodies

Contents

4 Push the envelope of innovation Comprehensive solutions for cancer research

5 From bench to bedside Translating discovery research into clinical therapy

6 Taking tumor tissue to task Tumor tissue dissociation

7 Tumor immunology Interactionbetweenimmunesystemandcancercells

8 The driving force of tumor development Cancer stem cells

9 Targeting cancer stem cells Tools for the enrichment and analysis of CSCs

10 Hematological tumors MultiplemyelomaandB-CLL

11 Tumor vascularization Endothelialcells,pericytes,andendothelialprogenitors

12 Spreading the tumor Circulating tumor cells

13 Capture circulating tumor cells Tools for enrichment and analysis of CTCs

14 Molecular analysis Expressionprofilingandmitochondrialenrichment

16 Optimal conditions Cell culture

17 Order information Placeyourorderbyfax,phone,oronline!

21 References Morethan13,000studiesusedMiltenyiBiotecproducts

Sorting out a cell separation strategyChoosing the optimal way of cell separation

Magnetic separation

Undesired cells are retained in a MACS Column placed in a MACS Separator.

The target cells pass through the column and are collected as the enriched, unlabeled cell fraction, depleted of non-targetcells.

Magnetic labeling

Non-targetcellsaremagnetically labeled withabiotinylatedantibody cocktail andAnti-BiotinMicroBeads.

Untouched isolation is performed by depletionofundesiredcells.Non-target cells are magnetically labeled and eliminated fromthecellmixture.Thenon-magneticallylabeled, untouched cell fraction contains the target cells. Formanydifferentcelltypes,MiltenyiBiotecoffers optimized MACS Cell Isolation Kits contain-ingpre-titratedcocktailsofantibodiesdirectedagainstnon-targetcells.

First magnetic labeling

Non-targetcellsaremagnetically labeled withabiotinylatedantibody cocktail and Anti-BiotinMicroBeads.

First magnetic separation

Undesired cells are retained in a MACS Column placed in a MACSSeparatorwhile the unlabeled cells pass through.

Second magnetic labeling

Target cells are magnetically labeled withMicroBeadsaccording to a subset marker.

Second magnetic separation

Target cells are retained in the column whileunlabeledcellspass through. After the column is removed from the separator, the target cells are eluted as the enriched, positively selected cell fraction.

Cell subsets can be isolated by first depleting the non-targetcellsandthenpositivelyselectingthecell subsets of interest. This strategy is useful if undesired cells in the cellsuspensionexpressthesameantigenthat is used for positive selection of the target cells.

Magnetic labeling

Cells of interest are magnetically labeled withMACSMicroBeads.

Elution of the labeled cell fraction

The column is removed from the separator. The retained cells are eluted as the enriched, positively selected cell fraction.

Magnetic separation

Cells are separated in a MACS Column placed in a MACS Separator.

Theflow-throughfraction can be collected as the negative fraction depleted of the labeled cells.

Positive selection means that the desired target cells are magnetically labeled and isolated as the magnetically retained cell fraction. Positive selection is the most direct and specific waytoisolatethetargetcellsfromaheterog-enouscellsuspension.BindingofMicroBeadsto the cell surface does not affect viability or functionofthecells.Bothfractions,labeledandunlabeled, can be recovered and used.

Positive selection Untouched isolationSequential sorting: Depletion followed by positive selection

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Push the envelope of innovationComprehensive solutions for cancer research

Streamline your experiments MiltenyiBiotecreagents,instruments,andservicesare designed to support scientists in achieving outstanding results. Fromsamplepreparation,throughcellsortingandculturetoperforming cell and molecular analyses.

MACS Sample Preparation

Thequalityofanexperimentstrictlydependsonthequalityof the sample preparation. Use the innovative gentleMACS™ Dissociatorforconsistent,fast,and gentle dissociation and homogenization of various tissues or cells in a closed system.

MACS Cell Separation

A large panel of MACS MicroBeadsandMicroBeadKitsare available for the isolation of virtually any cell type. The cells can be separated manually using MACS Magnets, or automaticallywiththeautoMACS® Pro Separator.

MACS Cell Analysis

We provide a large selection of monoclonal antibodies and kits for fluorescence microscopyandflowcytometry. The innovative MACSQuant® Analyzer is an extremelycompact,easy-to-usebenchtopflowcytometer. The instrument is fully automated, enabling absolute cell counting, and rare cell analysis.

MACS Cell Culture

The product portfolio for cell culture includes media aswellasrecombinantcytokinesandgrowthfactorsuptoGMPgrade. In addition, products for effective cell activation and expansionareavailable.

CliniMACS®

With the CliniMACS® Cell Separation System, Miltenyi Biotechassuccessfullytakenthe step from research into clinic.

MACSmolecular

We provide products for protein isolation and detection, mRNA purification andamplification,cDNAsynthesis and labeling, microRNAanalysis,aswellasmicroarray technologies and instrumentation.Alsoweoffergenomic services: gene and microRNAexpressionanalyses,array-CGH,andbioinformatics.

From bench to bedsideTranslating discovery research into clinical therapy

Researchers working for researchersAsapremierbiotechnologycompany,MiltenyiBioteciscommitted to the advancement of scientific understanding and medicine by providing products and services for biomedical research and cellular therapy.

In today’s research environment the translation of discovery research into efficacious clinical treatments is of utmost importance.Ourproductportfolioandvastinterdisciplinaryexpertisepaystributetothatfact.

A bridge from laboratory to clinicWeareawareofthemanychallengesfacedbytranslationalresearchers.Bridgingproof-of-principleresearchwith GMP-gradetherapiesisn'teasyandwecanhelpyou overcome these challenges.

DiscoverA portfolio of over 1,000 products is at your disposal, helping you to convert pioneering concepts into reality.

Advance Countlessresearchersworldwiderelyonourproductstoadvancetheirresearch;infact,morethan13,000peerreviewedscientific publications support this claim.

Translate Manyofourreagentsareavailableasresearch-gradeandGMP-gradeproducts,makingthetransitiontoclinicaltherapythatmucheasier.WealsomanufactureCE-markedinstrumentsand reagents for use in a clinical setting.

Discover. Advance. Translate.

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Taking tumor tissue to taskTumor tissue dissociation

Tissue dissociation that is reliable, reproducible, and rapidTheanalysisoftumorcellandstemcellpopulationsrequireseffectivemethodsfortissuedissociationfollowedby the isolation of viable cells.

Isolate viable cells from tumors with ease using the gentleMACS™ Dissociator Solidtumorsaremadeupofamixtureofcelltypeswhichareinterconnected to each other and surrounded by an extracellularmatrixcomposedofavarietyofproteinsandpolysaccharides.ThegentleMACS™Dissociatorreliablydissociatestheextracellularmatrixandcelladhesioncomponentswithoutharmingcellintegrity.

Specialized programs and protocols are optimized for tumor dissociation.Preparateyoursingle-cellsuspensionsfromvarious primary human or implanted mouse tumors using:

• gentleMACShumantumordissociationprograms

• gentleMACSmousetumordissociationprograms

• TumorDissociationKits

Automated dissociation of tissues assures that results are reproducible and reliable — all of that in a much shorter timeframe.

The gentleMACS™ Dissociator at a glance• Time-savingautomatedtissuedissociation

• Consistent,reliableresults

• Optimizedprogramsformultipleapplications

• Closedsystemenablingsterilesamplehandling

• Specificprogramsandprotocolsweredeveloped for cellular and molecular applications

Visit www.gentleMACS.comtolearnmoreabouthowthegentleMACSDissociatorcanadvanceyourresearch.

ThegentleMACSDissociatorandgentleMACSTubesaredesignedforefficient, reliable, and easy tissue dissociation.

TwotypesofgentleMACSTubesareavailable:CTubesandMTubes.CTubesgeneratesingle-cellsuspensionsforcellbiologyapplications(e.g.cellseparationandcellculture).MTubesallowforathoroughhomogenization of tissues or cells for molecular applications.

Tumor immunologyInteraction between immune system and cancer cells

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The immune system plays a crucial role in host protection against carcinogenesisbyeliminatingtumorcells.However,someofthetumor cells are not detected by the immune system and a variety of immunologic mechanisms contribute to tumor escape and

Tumor escape: Some tumor cells survive as they become insensitive to the elimination process due to genetic or epigenetic alterations. These cells continue to proliferate in an uncontrolled manner and more immune cells are attracted to the tumor site.

IL-4IL-5IL-10IL-13

MDSC:Myeloid-derivedsuppressorcell

CSC: Cancer stem cell

Tumor Lymphoid organ

Naive CD4+ T cell

Dendritic cell

TH2 cellTumor antigen

Dendritic cell

Circulating tumor cell

Dendritic cell

B cell

TH2 cell Treg cell

CD8+ T cell

IL-4IL-10IL-13TGF-β

iNKT cell

CD8+ T cell

MDSC

IL-10TGF-β

Macrophage

IL-6IL-10

Treg cell

MDSC

IL-10TGF-β

Macrophage

CD8+ T cell

Treg cell

IL-4

TGF-βIL-2

TGF-βIL-10

Monocyte

NK cell

CSC

Monocyte

subsequenttumorgrowthandangiogenesis.Webringyoua large array of tools to advance your research in tumor immunology, taking you from bench to bedside.

Findoutmoreat www.miltenyibiotec.com.

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Targeting cancer stem cellsTools for the enrichment and analysis of CSCs

Effective tools for cancer stem cell research IsolatecancerstemcellswitheaseusingourMicroBeadreagentsandkits,whichareoptimizedtoensureahighpurity and yield of target cells.

• Reliablyisolateviableandpurepopulationsofcancer stem cells from solid tumors and cancer cell lines.

• Isolatecellsonyourownschedulewhenever the sample becomes available.

• Targetanycellmarkerofyourchoice.

• Useenrichedsamplesfordownstreamapplications suchasflowcytometricormolecularanalysis.

MicroBeads: reagents for the isolation of cancer stem cellsMicroBeadsreagentscanbeuseddirectlyorincombinationfor the enrichment or depletion of defined cell markers:

• CD133MicroBeadKit1–8

• New:CD44MicroBeads

• New:CD24MicroBeadKit

• CD20MicroBeads

• CD15MicroBeads

• CD45MicroBeads

• CD271(LNGFR)MicroBeadKits

Target any cell type from any speciesFormaximumflexibility,indirectmagneticlabelingwithMACSMicroBeadsallowtheuseofanyprimaryantibodyforcell isolation. Monoclonal or polyclonal primary antibodies ofyourchoicecanbeeitherunconjugated,biotinylated,orfluorochrome-conjugated.

• Anti-FluorochromeMicroBeads

• Anti-BiotinMicroBeads

• Anti-IsotypeMicroBeads

• StreptavidinMicroBeads

CD+ cellswereisolatedfromamixtureofU(CD+)and(CD–)celllinesandthenisolatedusingtheCDMicroBeads,anLSColumn,andaMidiMACS™Separator.CellswerefluorescentlystainedwithCD-PEandanalyzedbyflowcytometryusingtheMACSQuantAnalyzer.

CD44-PE

Forw

ard

sca

tter

CD44-PE

Forw

ard

sca

tter

CD44+ CellsBefore separation

The driving force of tumor developmentCancer stem cells

Reinterpreting tumor biologyClassical tumor biology asserts that any malignantly transformed cell is immortal and may give rise tootherequallypotentcancerouscells.

Recent studies suggest that this hypothesis may not be the case, and that the clinical properties of human tumors may be the result of a small population of transformed stem cells withinthetumor—cancerstemcells.

Cancer stem cellsCancerstemcells(CSCs),alsoknownastumor-initiatingcells(TICs),arethoughttodrivetheprocessoftumorigenesisonthebasisofself-renewalandthegenerationofanaberranttumorcelluponCSCdivision.CSCswouldthereforeeffectuatetumormetastasis and tumor relapse; a theory that is contrary to classical tumor biology hypotheses.

Possible implications of this hypothesis:• MetastasesaremediatedbyCSCsandnot any tumor cell.

• CSCsareresistanttoconventionalchemotherapies and facilitate tumor relapse.

AbetterunderstandingofCSCpathogenesiswouldnotonlyserve to improve diagnostic procedures, but also to develop therapies that specifically target these cells, hindering tumor recurrence.

ReadfurthertodiscoverhowMiltenyiBioteccanhelpyou make strides in cancer research or visit: www.miltenyibiotec.com/csc.

Tumor type Cell surface marker

Acutemyeloidleukemia(AML)35 CD34+/CD38–

Breastcancer36 ESA+/CD44+/CD24–/Lineage–

Ovariancancer37–39 CD133+

CD44+/CD117+

CD24+

Glioblastoma*40–42 CD133+

CD15+

Medulloblastoma40,41,43 CD133+

CD15+

Smallcellandnon-smallcell lung cancer44

CD133+

Hepatocellularcarcinoma45 CD45–/CD90+

Prostate cancer⁵ CD44+/A2B1hi/CD133+

Colon cancer6,46–49 CD133+

CD44+

CD26+

Melanoma50–53 CD20+

ABCB5+

CD271+

Pancreas adenocarcinoma54 CD44+/CD24+/EpCAM+

Renal carcinoma55 CD133enhancesvascularization

Headandnecksquamouscellcarcinoma(HNSCC)56

CD44+

*CDexpressionmaybeaffectedbycellcultureconditions.Cell surface markers of cancer stem cells in different types of tumors.Forarespectiveproductlistpleaserefertopages–.

CD-CD+cellswereisolatedfromCMLcelllineKZ.CD+cellswerefirstdepletedusingtheCDMicroBeadKitandthenpositivelyselectedforCDusingtheCDMicroBeads.CellswerefluorescentlystainedwithCD-APCandCD-FITCandanalyzedbyflowcytometryusingtheMACSQuant Analyzer.

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Positive fraction

CD24-FITC

CD

44-A

PC

Original fraction

CD24-FITC

CD

44-A

PC

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Hematological tumorsMultiple myeloma and B-CLL

High performance in hematologicalcell enrichment and analysisMiltenyiBioteccontinuestoexpandonasignificantportfolioof MACS Products for hematological research. These include products for the investigation of normal hematopoiesis and reagents for the study of hematological malignancies.

Leukemic stem cells It has been proposed that transformation of hematopoietic stemcellsresultsinthegenerationofleukemicstemcells(LSCs)—tumorcellswithuncontrollableself-renewalcapabilities.

In order to elucidate the pathogenic processes behind this transformation, many researchers are currently focusing on theidentification,enrichment,andcharacterizationofLSCs.

AccuratelyisolateLSCswithinminutes:

• CD34MicroBeadKit⁹¹⁰

• CD34MultiSortKit¹¹

• Specialprotocoldevelopedfor isolationofCD34+/CD38–cells¹²

Multiple myelomaIsolateCD138+cellswithminimumhands-ontimeandamaximumyieldoftargetcells,evenfromweaklyinfiltratedmyeloma patient samples.

• CD138MicroBeads¹³¹⁴

• WholeBloodCD138MicroBeads

Investigating Hodgkin’s lymphoma ThehallmarkindicatorofHodgkin'slymphomaistheidentificationofCD30+/CD15+Reed-Sternbergcells.These rare and highly fragile cells can be enriched using:

• CD30MicroBeads

Chronic lymphocytic leukemia Bcellchroniclymphocyticleukemiaisoneofthemostcommontypesofleukemia.IsolationofuntouchedBcellscanbeachievedfromtumorcell-containingsamplesusing:

• BCellIsolationKit(B-CLL)

Isolated iNKT+ cells


Before separation

Before separation

FlowcytometricanalysisofCD+ cells isolated from peripheral blood mononuclearcells(PBMCs)usingtheCDMicroBeadKit,anMSColumn,and a MiniMACS™ Separator.

CD138+plasmacellswereisolatedfromhumanPBMCsusingCD20andCD138MicroBeads,anLDandtwoMSColumns,andappropriateMACSSeparators.CellsarefluorescentlystainedwithCD138-PEandCD19-APC.

CD34+ cells

CD138+ cells

Before separation

Before separation

CD34-PE

CD45

-FITC

CD138-PE

CD19

-APC

CD34-PE

CD45

-FITC

CD138-PE

CD19

-APC

Tumor vascularizationEndothelial cells, pericytes, and endothelial progenitors

Novel tools to assess tumor vascularizationVascularization of a developing solid tumor is considered to be akeystepintumorgrowth,invasionandmetastasis.Newvesselformation involves the recruitment of endothelial progenitor cells(EPCs)frombonemarrow,resultinginanincreaseofEPClevelsintimesofsignificanttumorgrowth.

Using EPCs to evaluate tumor progression Growingevidencesuggeststhattheefficacyofanti-angiogenictherapiescanbequantitatedbyenumeratingcirculatingEPCs.Moreover,EPCnumberscouldalsoindicatetumorprogression.

TaketheconvenientrouteforEPCanalysisusingtheEPCEnrichmentandEnumerationKit:

• Significantlyreducesyouranalysistimebyflowcytometry

• OptimizedforusewiththeMACSQuantAnalyzerfor walk-awaysampleprocessing,flowcytometricgating, andEPCenumeration(comingsoon)

EPCsaredefinedbytheexpressionofCD34,CD133,andCD309and are considered to be a parameter for assessing a number of diseases involving vascular repair.

Stem cells making a niche for themselves Recentreportssuggesttheexistenceofperivascular–cancerstemcellniches;microenvironmentswhereendothelialcellsandpericytesinteractcloselywithself-renewingCSCs.

WehavedevelopedspecificMicroBeadsfortheefficientenrichment of endothelial cells and pericytes:

• CD146MicroBeadKit

• CD105MicroBeads

• CD31MicroBeadKit



Before separation

Before separation

CD+CD+CD(VEGFR-/KDR)+ EPCswereidentifiedandenumeratedfrom a leukocyte sample and analyzed using the MACSQuant Analyzer.

CD+cellswereseparatedfromlipoaspirateusingtheCDMicroBeadKit,anLSColumn,andaMidiMACSSeparator.CellswerestainedwithCD-APCandCD-FITC.

CD34+ cell fraction

CD146+ cell fraction

Before separation

Before separation

CD133/2 (293C3)-PE

CD

34-F

ITC

R4

CD133/2 (293C3)-PE

CD

309

(VEG

FR-2

/KD

R)-

AP

C

R5

CD146-APC

CD34

-FITC

CD146-APC

CD34

-FITC

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Spreading the tumorCirculating tumor cells

Unravelling the mechanisms of metastasisCirculatingtumorcells(CTCs)arecellsthatareshedfromtheprimarytumorandenterthecirculationwheretheycanspreadto anatomical sites distant from the primary tumor and form metastases.

Towards a better understanding of circulating tumor cellsCirculatingtumorcellsarefoundatextremelylowfrequencies,oftenatthedetectionlimitofmanyanalyticaltechniques. This places a great challenge on their identification and characterization.Toovercomethis,pre-enrichmentof rare CTCs is necessary.

Optimize the detection and analysis of circulating tumor cellsCirculating tumor cells can be easily enriched usingthefollowingmarkerspriortoanalysis:

• CD326(EpCAM)

• ErbB-2(HER2)

• Anti-Melanoma(MCSP,NG2)

• CD45

• Anti-Cytokeratines

Retention of target cell population in a column.

Magnetic labeling of a specific cell population usingMACSMicroBeads.

1. Elutionoftargetcells.

2. FixationbyaddingInsideFix.

1.Applicationofthefixedcells onto a second column.

2. Permeabilizationwith Inside Perm.

3. Application of staining reagents onto the column.

Elution,substrateincubationandflowcytometricorimmunocytochemical evaluation.

In-columnintracellularstainingofCTCsisolatedusing the MACS Technology

Capture circulating tumor cellsTools for enrichment and analysis of CTCs

Enrich circulating tumor cells for discovery researchMACSMicroBeadscanbeusedfortheisolation of circulating tumor cells from a variety of sources.

• Peripheralbloodmononuclearcells(PBMCs)

• Bonemarrow

• Lymphoidtissue

• Peripheralbloodbuffycoats

Consistent quality in CTC enrichmentUseoneofthefollowingMicroBeadReagentsfortheconvenient enrichment of circulating tumor cells:

• CD326(EpCAM)MicroBeads21–27

• ErbB-2(HER2)MicroBeads23

• CarcinomaCellEnrichmentKit28

• Anti-Melanoma(MCSP)Microbeads29,30

• CD45MicroBeads31–33

Refer to www.miltenyibiotec.com/tumorcells for more details.

Achieve paramount performance in circulating tumor cell detectionAnalysis of circulating tumor cells is inherently limited by the sensitivityofthedetectionsystem.OvercometheselimitationswithMACSEnrichmentandDetectionKits.

• Circulatingtumorcellsareenriched using the desired cell marker.

• Enrichedcellscanbedirectlystainedwhile in the column minimizing cell loss.

• Stainedcellsareelutedandcanbe immedately used for cell analysis.

A complete kit for enrichment and detectionThefollowingEnrichmentandDetectionKitshavebeendesigned for optimal detection of circulating tumor cells:

• CD326(EpCAM)TumorCellEnrichmentandDetectionKit

• ErbB-2TumorCellEnrichmentandDetectionKit

• CarcinomaCellEnrichmentandDetectionKit¹⁶-²⁰

• MelanomaCellEnrichmentandDetectionKit

Forintracellularstainingofcancerstemcells:

• InsideStainKit

• Anti-Cytokeratinantibodies

Immunocytochemical detection of breast cancer cells enriched using theCarcinomaCellEnrichmentandDetectionKitandstainedwith Anti-Cytokeratin-FITCandAnti-FITC-AlkalinePhosphataseplussubstrate.

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Molecular analysisExpression profiling and mitochondrial enrichment

Excel in tumor molecular analysesMACSmolecular provides a highly innovative range of products andserviceswithastrongfocusonmicroRNAandgeneexpressionprofiling.

Convenient microarray services for cancer researchForlaboratorieswhohavenoorlimitedaccesstomicroarraytechnologiesweofferacompletemicroarrayservice:fromsample preparation to the generation of a comprehensive bioinfomatics report.

WeareanofficallycertifiedAgilentServiceProviderwithover10yearsexperienceinthisfield.

Ourhigh-qualityAgilentMicroarrayServices—fromRNAextractiontocomprehensivebioinformaticservices—resultinready-to-publishdata.

Microarray analysis couldn't be easier• SendyoursamplestoMiltenyiBiotec.

• Sampleprocessingandanalysiswillbeperformed usingstringentqualitycontrolstandards.

• Ourin-housebioinformaticsteamwillcompile a clear and comprehensive report of your data, including arecordofallexperimentalstepsanddataanalyses.

µMACS™ SuperAmp Technology for single cell analysis µMACS™SuperAmpTechnologyenablesgeneexpressionprofilingdowntoasinglecell.ThesourceRNAcanbederivedfrom:

• cellssortedwithMACSTechnology

• laser-captured,microdissectedcells

• cellssortedbyflowcytometry

• tissuebiopsies

• 1–10,000cells

Reverse-complementary miR-27a sequence and synthetic variants miR-27a GCGGAACTTAGCCACTGTGAA mut1 GCGGAACTTACCCACTGTGAA mut2 GCGGACCTTACCCACTGTGAA

Probe-targetspecificityofmiRXploreMicroarray

Hepatocellular carcinoma cell RNA

Rela

tive

sign

al in

tens

ity

(%)

Glioblastoma cell RNA

Hippocampus RNA

CD4+ T cell RNA

100

miR -27a miR -27a - mut1 miR -27a - mut2

90

80

70

60

50

40

30

20

10

0

Rel

ativ

e si

gn

al in

ten

sity

(%)

miR-465A-5P

100

90

80

70

60

50

40

30

20

10

0

miR-465B-5P miR-465C-5P miR-465D

miR-465 family miR-465A-5PUAUUUAGAAUGGCACUGAUGUGAmiR-465B-5PUAUUUAGAAUGGUGCUGAUCUGmiR-465C-5PUAUUUAGAAUGGCGCUGAUCUGmiR-465D UAUUUAGAAUGGUACUGAUGUG

SpecificdetectionofmiR-465familymembers

State-of-the-art microRNA expression profilingUseoneofthehigh-qualitymiRXplore™MicroarrayKits—reliabletoolsforextensivemicroRNAexpressionprofiling.

• Expertcoverageofsequences

• HighsensitivemicroRNAprofiling

• Excellentspecificity

• Consistentdata

• Sampleindependentnormalization

The faster approach to mitochondria enrichmentMany studies have described mitochondrial abnormalities in tumor tissues and cancer cell lines. Investigators often rely on density centrifugation for mitochondrial enrichment—a time-consumingandlaborioustechnique.

BenefitfromtheMitochondriaIsolationKit:afaster and simplier procedure that is also more accurate.

• Highyieldsofisolatedmitochondria

• Maintainsorganelleintegrity

• Highpuritylevels

• Easyandstraightforwardoperation

• Size-independentisolation

Forfurtherinformationaboutourmolecularservices, visit www.miltenyibiotec.com.

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Technical support for experimental design and microarray selection• microRNAmiRXploreMicroarrays• AgilentWholeGenomeMicroarrays

RNA extraction and quality control

Optional:•Amplification and quality control•SuperAmp Service1: 1–10,000 cells

Synthesis and purification of fluorescently labeled probes

Microarray hybridization

Image capture and analysis of primary data

Optional: Bioinformatics Services2 •ClusterAnalysis•DiscriminatoryGenesAnalysis•PathwayAnalysis

Results and reportDataonCD-ROM

1miRNAscannotbeamplifiedwiththeSuperAmpService. 2PleaseinquireformicroRNABioinformaticsServices.

Send sample—receive results

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Optimal conditionsCell culture

Comprehensive solutions for cell culture

Refined culture conditions for cancer cellsWeofferanexpandingportfolioofprovenMACS®andStemgent® Media for the reliable culture of cancer cells.

• Ready-to-use

• Broadrangeofapplications

• Easy-to-followprotocols

• Manufacturedtothehigheststandards

Benefit from an array of cytokines and growth factorsOurcytokinesandgrowthfactorsaredevelopedforoptimalperformance in cell culture and bioassays. The cytokine productsareactivity-testedtoensurethemostfunctionallyactive biological results possible.

• Stringentlytestedforpurity,endotoxinlevels,andstructural homogeneity

• Reducingtheconcernthatco-purifiedcontaminantswill negatively impact cell culture behavior

• Research-,premium-,andGMP-gradeproducts

Visit www.miltenyibiotec.com/Cytokines for a comprehensive list of available cytokines.

WIT Media*Cells derived from normal breast epithelial tissue represent an importantmodelforcancerstudies.Breastprimaryepithelialcells(BPECs)thatarenotmaintainedonfeedercellsundergoM0arrestwithinafewpopulationdoublings.

With Stemgent’s WIT Media products, breast epithelial cells are cultured under conditions that maintain propagation through many passages, and culture a cell type that is much more representative of that found in human breast cancers.

• Definedandserum-free

• Bovinepituitaryextract–free

• Feeder-free

• Optimallysupportthecultureofhumanprimary, immortalized, and transformed breast epithelial cells

*MiltenyiBiotecistheauthorizeddistributorforStemgent,Inc.ThisproductisnotdistributedbyMiltenyiBiotecintheUSA.

Order informationPlace your order by fax, phone, or online!

Sample preparationProduct Description Order no.

gentleMACS™ Dissociator

gentleMACS Starting Kit Benchtopinstrumentfor the automated dissociation or homogenization of tissues

130-093-235

gentleMACS™ Tubes

C Tubes gentleMACS Tubes for the dissociation of tissues to obtain single-cellsuspen-sions

130-093-237

M Tubes gentleMACS Tubes for the homogenization of tissues or cells to isolate biomolecules

130-093-236(25tubes)130-093-458(50tubes)

MACS® Pre-Separation Filters

Pre-SeparationFilters,30 µm

Filtersfortheremovalof cell clumps

130-041-407

Pre-SeparationFilters,70 µm

Filtersfortheremovalof large cell clumps

130-095-823

MACS® Reagents for sample preparation

DeadCellRemovalKit Depletionofdeadcells

130-090-101

RedBloodCellLysisSolution(10×)

Lysisoferythrocytes 130-094-183

Cell separation reagentsProduct Description Order no.

Cancer stem cells

CD133MicroBeadKit,human

Positive selection 130-050-801

IndirectCD133MicroBeadKit,human


CD44MicroBeads,human Positive selection 130-095-194


DepletionofCD24+ cells

130-095-951

CD20MicroBeads,human

Positive selectionor depletion

130-091-104

CD271(LNGFR)MicroBeadKits


130-092-819(PE)130-092-283(APC)



130-046-601

Circulating tumor cells (epithelial tumor cells)

CD326(EpCAM)MicroBeads,human


GrowthcurveofimmortalizedbreastprimaryepithelialcellsinWIT-IMedium

Medium Description

WIT-P* KeepsBPECsgrowinginculturewellbeyondthestageswheregrowtharrestandsenescenceareobservedwithotherculturemedia.

WIT-I* Allowsthein vitro propagation of human breast epithelialcellsimmortalizedfromBPECsculturedinWIT-PMedium,andalsomaintainstheirluminalepithelial character.

WIT-T* OptimizedforcellsthathavebeentransformedfromWIT-PculturedBPECs,andalsomaintainstheirluminal epithelial character.

0,0

2,0

4,0

6,0

8,0

10,0

12,0

0 10 20 30 40

Days

MorphologyofimmortalizedbreastepithelialcellsinWIT-IMedium

Population doublings BPE

Pop

ulat

ion

dou

blin

gs

Days

WIT-I

Product Description Order no.

CD326(EpCAM)MicroBeads+CD326(EpCAM)-PE,human

Positive selection and staining

130-092-234

CD326(EpCAM)TumorCellEnrichmentandDetectionKit, human


130-090-500

Anti-ErbB-2MicroBeads,human


ErbB-2TumorCellEnrichmentandDetectionKit,human


130-090-499

CarcinomaCellEnrichmentKit, human


CarcinomaCellEnrichmentandDetectionKit–Immunocytochemistry, human


130-060-301

Melanoma cells

Anti-Melanoma(MCSP)MicroBeads,human

Positive selection of human melanoma cells

130-090-452

MelanomaCellEnrichmentandDetectionKit,human

Enrichmentandimmunocytochemical detection of melanoma cells

130-090-523

Depletion of leukocytes

CD45MicroBeads,human Enrichmentofhumantumor cells by depletion of leukocytes

130-045-801

WholeBloodCD45MicroBeads,human


Hematopoietic tumor cells


Positive selection 130-046-702130-046-703

IndirectCD34MicroBeadKit,human


CD34MultiSortKit,human




130-092-263



130-051-301

CD138MicroBeads+CD138-PE,human


130-090-503

WholeBloodCD138MicroBeads,human


BCellIsolationKit(B-CLL),human

Depletionofnon-Bcells 130-093-660

18 19

Molecular applicationsProduct Order no.

μMACS mRNA Starting Kit 130-075-202

μMACSOne-stepcDNAStartingKit 130-091-989

μMACS SuperAmp Starting Kit

130-093-251

SuperAmp Service 160-000-936

AgilentWholeHumanGenomeMicroarrayService4×44K,Two-color

160-001-081

AgilentWholeMouseGenomeMicroarrayService,4×44K,Two-color

160-001-082

AgilentWholeRatGenomeMicroarrayService,4×44K,Two-color

160-001-078

AgilentHumanGenomeCGHMicroarrayService4×44K

160-000-966

AgilentMouseGenomeCGHMicroarrayService4×44K

160-000-964

AgilentHighDefinition-CGHCustomMicroarrayService

160-000-965

miRXploreMicroarrayService 160-001-143

miRXploreMicroarrayUniversalReferenceService(UR)

160-001-161

Mitochondria MidiMACS Starting Kit, human 130-094-872

Mitochondria QuadroMACS Starting Kit, human 130-094-833

Indirect magnetic labeling reagentsProduct Description Order no.

Anti-Fluorochrome MicroBeads

Anti-FITCMicroBeads Positive selection or depletion

130-048-701

Anti-FITCMultiSortKit Magnetic separation of cells according to multiple surface markers

130-058-701

Anti-PEMicroBeads Positive selection or depletion

130-048-801

Anti-PEMultiSortKit Magnetic separation of cells according to multiple surface markers

130-090-757

Anti-APCMicroBeads Positive selection or depletion

130-090-855

Anti-APCMultiSortKit Magnetic separation of cells according to multiple surface markerst

130-091-255

Anti-Cy5/Anti-AlexaFluor647MicroBeads


130-091-395

Anti-Cy7MicroBeads Positive selectionor depletion

130-091-652

Anti-Biotin MicroBeads

Anti-BiotinMicroBeads Positive selection or depletion

130-091-255

Anti-BiotinMultiSortKit Magnetic separation of cells according to multiple surface markers

130-091-395

Cell analysis reagentsProduct FITC PE APC Biotin VioBlue pure PerCP

Cancer stem cells

CD133/1(W6B3C1) - - - - - 130-092-395 -

CD133/1(AC133) - 130-080-801 130-090-826 130-090-664 - 130-090-422 -

CD133/2(293C3) - 130-090-853 130-090-854 130-090-852 - 130-090-851 -

CD133/2CE(293C3) - 170-070-702 - - - - -

CD133/2(AC141) - 130-080-901 - - - 130-090-423 -

CD44 130-095-195 130-095-180 130-095-177 - - - -

CD24Coming 2010

CD20 130-091-108 130-091-109 - - 130-094-167 - 130-094-976

Circulating tumor cells / epithelial tumor cells

CD326(EpCAM) 130-080-301 130-091-253 130-091-254 - - - -

Circulating tumor cells / epithelial tumor cells

Anti-Melanoma(MCSP,NG2) - 130-091-225 130-091-252 - - - -

Leukocytes

CD45 130-080-202 130-080-201 130-091-230 - 130-092-880 - 130-094-975

Cell separation reagents


Tumor cell detection kits

CD326(EpCAM)TumorCellEnrichmentandDetectionKit

Positive selection and staining 130-090-500

ErbB-2TumorCellEnrichmentandDetectionKit Positive selection and staining 130-090-499

CarcinomaCellDetectionKit Detectionofcytokeratin-expressinghumanepithelialtumorcells 130-090-463

CarcinomaCellEnrichmentandDetectionKit–Immunocytochemistry

Positive selection and staining 130-060-301

MelanomaCellEnrichmentandDetectionKit Enrichmentandimmunocytochemicaldetectionofmelanomacells 130-090-523

Product FITC PE APC Biotin VioBlue pure PerCP

Intracellular staining

Anti-Cytokeratin(CK3-3E4) - - - - - 130-090-865 -

Anti-Cytokeratin(CK3-6H5) 130-080-101 - - - - 130-090-866 -

Anti-Cytokeratin-AlkalinePhosphatase

- - 130-090-462 - - - -



Streptavidin MicroBeads

Streptavidin MicroBeads

Positive selection or depletion

130-048-102

Monoclonal anti-Ig MicroBeads

Anti-MouseIgG1MicroBeads


130-047-102

Anti-MouseIgG2a+bMicroBeads


130-047-202

Anti-MouseIgMMicroBeads


130-047-302

Anti-RatKappaMicroBeads


130-047-401

Anti-IgGMicroBeads,human


130-047-501


CD30MicroBeads,human Positive selection 130-051-401

CD30MicroBeads+CD30-PE,human


LineageCellDepletionKit, human

Untouched cell enrichment

130-092-211

Endothelial cells



130-091-935



130-051-201



130-093-596

1918

20

21

Product Description Capacity / components Order no.

Intracellular staining

Inside Stain Kit Fixationandpermeabilizationofcellsforintracellularstainingwithsmallstainingvolumesandminimal cell loss

upto50tests 130-090-477

FcRBlockingReagent,human BlockingofFcreceptor-mediatedbindingtohuman/non-humanprimatecells

2mL 130-059-901



CD34 130-081-001 130-081-002 130-090-954 - - - -

CD38 130-092-259 130-092-260 130-092-261 130-092-288 - - -

CD138 - 130-081-301 130-091-250 - - - -



MCCD34StemCellCocktail Forcontrolstainingofhumanstemcells 50tests 130-093-427


Endothelial cells

CD31 130-092-654 130-092-653 130-092-652 - - - -

CD105 - 130-094-941 130-094-926 130-094-916 - - -

CD146 130-092-851 130-092-853 130-092-849 130-092-852 - 130-092-850 -

CD309(VEGFR-2/KDR) - - 130-093-601 130-093-603 - - -


Endothelial cells

EPCEnrichmentandEnumerationKit Enumerationofendothelialprogenitorcells 20tests⁶ 130-093-477

ReferencesMore than 13,000 studies used Miltenyi Biotec Products

Xiao-Ling,L.12. et al.(2007)Differentialgeneexpressioninhumanhematopoieticstemcellsspecifiedtowarderythroid, megakaryocytic, and granulocytic lineage. J.Leukoc.Biol.82:986–1002.

Aldo,M.(2009)MicroRNAs15aand16regulatetumor13. proliferationinmultiplemyeloma.Blood113:6669–6680.

Chen,L.14. et al.(2010)Identificationofearlygrowthresponseprotein1(EGR-1)asanoveltargetforJUN-inducedapoptosisinmultiplemyeloma.Blood115:61.

Igreja,C.(2007)Characterizationandclinicalrelevanceof15.circulatingandbiopsy-derivedendothelialprogenitorcellsinlymphomapatients.Haematologica92:433–434.

Dome,B.16. et al.(2006)Identificationandclinicalsignificanceofcirculatingendothelialprogenitorcellsinhumannon-small cell lung cancer. Cancer Res. 66: 7341–7347.

Molnar,B.17. et al.(2001)Circulatingtumorcellclustersintheperipheral blood of colorectal cancer patients. Clin. Can. Res.7:4080–4085.

Campos, M. 18. et al.(2008)Phenotypicandgeneticcharacterization of circulating tumor cells by combining immunomagneticselectionandFICTIONtechniques. J.Histochem.Cytochem.56:667–675.

Molnar,B.(2001)Circulatingtumorcellclustersinthe19. peripheral blood of colorectal cancer patients. Clin. Cancer Res.7:4080–4085.

Schmidt,H.20. et al.(2006)Asynchronousgrowthofprostatecancer is reflected by circulating tumor cells delivered from distinct, even small foci, harboring loss of heterozygosity of thePTENgene.CancerRes.66:8959–8965.

Serrano, M. 21. et al.(2007)Persistenceofcirculatingtumourcells after treatment predicts clinical outcome in breast cancerpatients.ASCOAnnualMeetingProceedings(Post-MeetingEdition).J.Clinic.Onco.25;18S:21080.

Morgan, T.M. 22. et al.(2009)Disseminatedtumorcellsinprostate cancer patients after radical prostatectomy andwithoutevidenceofdiseasepredictsbiochemicalrecurrence.Clin.CancerRes.15:677–683.

Rotella, S. 1. et al. (2009)Cellswithcharacteristicsofcancerstem/progenitorcellsexpresstheCD133antigeninhumanendometrialtumors.Clin.CancerRes.15:4299–4311.

Botchkina,I.2. et al.(2008)TheroleofCD133innormalhumanprostatestemcellsandmalignantcancer-initiatingcells. Cancer Res. 68: 9703–9711.

Xu,M.3. et al.(2008)MonoclonalantibodyCC188bindsacarbohydrateepitopeexpressedonthesurfaceofbothcolorectalcancerstemcellsandtheirdifferentiatedprogeny. Clin. Cancer Res. 14: 7461–7469.

Vermeulen,L.4. et al.(2008)Single-cellcloningofcoloncancerstemcellsrevealsamulti-lineagedifferentiationcapacity. Proc. Natl. Acad. Sci. USA 36: 13427–13432.

Collins, A. T. 5. et al.(2005)Prospectiveidentificationoftumorigenicprostatecancerstemcells.CancerRes.65:10946–10951.

Dalerba,P.6. et al.(2007)Phenotypiccharacterizationofhuman colorectal cancer stem cells. Proc. Natl. Acad. Sci. USA104:10158–10163.

Kemper, K. 7. et al.(2010)TheAC133epitope,butnottheCD133protein,islostuponcancerstemcelldifferentiation.Cancer Res. 70: 719–29.

Nikolaos,A.(2009)Chemoresistantcolorectalcancercells,8. the cancer stem cell phenotype, and increased sensitivity toinsulin-likegrowthfactor-Ireceptorinhibition.CancerRes.69:1951–1957.

Rizo, A. 9. et al.(2009)RepressionofBMI1innormalandleukemichumanCD34+cellsimpairsself-renewalandinducesapoptosis.Blood114:1498–1505.

Yoshimoto,G.10. et al.(2009)FLT3-ITDup-regulatesMCL-1topromote survival of stem cells in acute myeloid leukemia viaFLT3-ITD-specificSTAT5activation.Blood114: 5034–5043.

Schiedlmeier,B.(2002)Quantitativeassessmentof11. retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficientmice.Blood95:1237–1248.


Cell analysis reagents

22

Witzig,T.E.23. et al.(2002)Detectionofcirculatingcytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin. CancerRes.8:1085–1091.

Molloy, T. J. 24. et al.(2008)Towardsanoptimizedplatformforthedetection,enrichment,andsemi-quantitationcirculatingtumorcells.BreastCancerRes.Treat.112:297–307.

Zou,G-M.25. et al.(2008)Small-moleculeinhibitoroftheAPendonuclease1/REF-1E3330inhibitspancreaticcancercellgrowthandmigration.Mol.CancerTher.7:2012–2021.

Dmitri,B.26. et al.(2006)Antibodytargetingoflong-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models. Cancer Res. 66: 6732–6740.

Fehm,T.27. et al.(2002)Cytogeneticevidencethatcirculatingepithelialcellsinpatientswithcarcinomaaremalignant.Clin. Cancer Res. 8: 2073–2084.

Thomas,E.(2002)Detectionofcirculatingcytokeratin-28. positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin. CancerRes.8:1085–1091.

Molnar,B.29. et al.(2001)CirculatingTumorCellClustersinthePeripheralBloodofColorectalCancerPatients.Clin.CancerRes.7:4080–4085.

Ulmer. A. 30. et al.(2004)Immunomagneticenrichment,genomic characterization, and prognostic impact of circulatingmelanomacells.Clin.CancerRes.10:531–537.

Ulmer, A. 31. et al.(2008).Visualizationofcirculatingmelanomacellsinperipheralbloodofpatientswithprimaryuvealmelanoma. Clin. Cancer Res. 4: 4469–4474.

Bluemke,K.(2009)Detectionofcirculatingtumorcells32. inperipheralbloodofpatientswithrenalcellcarcinomacorrelateswithprognosis.Can.Epidemiol.BiomarkersPrev.18: 2190–2194.

Morgan, T.M. 33. et al.(2009)Disseminatedtumorcellsinprostate cancer patients after radical prostatectomy andwithoutevidenceofdiseasepredictsbiochemicalrecurrence.Clin.CancerRes.15:677–683.

23

Thomas,E.(2002)Detectionofcirculatingcytokeratin-34. positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy. Clin. CancerRes.8:1085–1091.

Bonnet,D.andDick,J.E.(1997)Humanacutemyeloid35.leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 3: 730–737.

Al-Hajj,M.36. et al. (2003)FromtheCover:Prospectiveidentificationoftumorigenicbreastcancercells.Proc.Natl.Acad. Sci. USA 100: 3983–3988.

Ferrandina,G.37. et al. (2009)CD133antigenexpressioninovariancancer.BMCCancer.7:221.

Zhang,S.38. et al.(2008)Identificationandcharacterizationofovariancancer-initiatingcellsfromprimaryhumantumors.Cancer Res. 68: 4311–4320.

Gao,M-Q.39. et al.(2010)CD24þcellsfromhierarchicallyorganized ovarian cancer are enriched in cancer stem cells. Oncogene29:2672–2680.

Singh, S.K. 40. et al. (2003)Identificationofacancerstemcellinhumanbraintumors.CancerRes.63:5821–5828.

Singh, S.K. 41. et al. (2004)Identificationofhumanbraintumour initiating cells. Nature 432: 396–401.

Son, M. J. 42. et al.(2009)SSEA-1isanenrichmentmarkerfortumor-initiatingcellsinhumanglioblastoma.CellStemCell4:440–452.

Read,T-A.43. et al. (2009)IdentificationofCD15asamarkerfortumor-propagatingcellsinamousemodelofmedulloblastoma.CancerCell15:135–147.

Eramo,A44. et al. (2008)Identificationandexpansionofthetumorigeniclungcancerstemcellpopulation.CellDeathDiffer.15:504–514.

Yang,Z.F.45. et al. (2008)Identificationoflocalandcirculatingcancerstemcellsinhumanlivercancer.Hepatology47:919–928.

O'Brien,C.A.46. et al.(2007)Ahumancoloncancercellcapableofinitiatingtumourgrowthinimmunodeficientmice.Nature445:106–110.

Ricci-Vitiani,L.47. et al.(2007)Identificationandexpansionofhumancolon-cancer-initiatingcells.Nature445:111–115.

Du,L.48. et al. (2008)CD44isoffunctionalimportanceforcolorectalcancerstemcells.Clin.CancerRes.14:6751.

Pang, R. 49. et al.(2010)AsubpopulationofCD26+ cancer stem cellswithmetastaticcapacityinhumancolorectalcancer.CellStemCell6:603–615.

Fang,D.50. et al.(2005)Atumorigenicsubpopulationwithstemcellpropertiesinmelanomas.CancerRes.65:9328–9337.

Kamstrup, M.R. 51. et al.(2007)Putativecancerstemcellsincutaneousmalignancies.Exp.Dermatol.16:297–301.

Schatton, T. 52. et al. (2008)Identificationofcellsinitiatinghumanmelanomas.Nature45:doi:10.1038/nature06489.

Boiko,A.53. et al.(2010)Humanmelanoma-initiatingcellsexpressneuralcrestnervegrowthfactorreceptorCD271.Nature 466: doi:10.1038/nature09161.

Li,C.54. et al.(2010)Identificationofpancreaticcancerstemcells. Cancer Res. 67: 1030–1037.

Bruno,S.55. et al.(2006)CD133+ renal progenitor cells contribute to tumor angiogenesis. Am. J. Pathol. 169: 2223–2235.

Prince,M.E.56. et al.(2007)Identificationofasubpopulationofcellswithcancerstemcellpropertiesinheadandnecksquamouscellcarcinoma.Proc.Natl.Acad.Sci.USA104:973–978.

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