Cancer Research Biotec
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Transcript of 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
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
Isolated iNKT+ cells
Isolated iNKT+ cells
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
Positive selection 130-091-895
CD44MicroBeads,human Positive selection 130-095-194
CD24MicroBeadKit,human
DepletionofCD24+ cells
130-095-951
CD20MicroBeads,human
Positive selectionor depletion
130-091-104
CD271(LNGFR)MicroBeadKits
Positive selectionor depletion
130-092-819(PE)130-092-283(APC)
CD15MicroBeads,human
Positive selectionor depletion
130-046-601
Circulating tumor cells (epithelial tumor cells)
CD326(EpCAM)MicroBeads,human
Positive selection 130-061-101
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
Positive selection and staining
130-090-500
Anti-ErbB-2MicroBeads,human
Positive selection 130-090-482
ErbB-2TumorCellEnrichmentandDetectionKit,human
Positive selection and staining
130-090-499
CarcinomaCellEnrichmentKit, human
Positive selection 130-060-101
CarcinomaCellEnrichmentandDetectionKit–Immunocytochemistry, human
Positive selection and staining
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
Positive selection 130-090-872
Hematopoietic tumor cells
CD34MicroBeadKit,human
Positive selection 130-046-702130-046-703
IndirectCD34MicroBeadKit,human
Positive selection 130-046-701
CD34MultiSortKit,human
Positive selection 130-056-701
CD38MicroBeadKit,human
Positive selectionor depletion
130-092-263
CD138MicroBeads,human
Positive selectionor depletion
130-051-301
CD138MicroBeads+CD138-PE,human
Positive selectionor depletion
130-090-503
WholeBloodCD138MicroBeads,human
Positive selection 130-093-062
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
Positive selectionor depletion
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
Product Description Order no.
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 - - - -
Order informationPlace your order by fax, phone, or online!
Product Description Order no.
Streptavidin MicroBeads
Streptavidin MicroBeads
Positive selection or depletion
130-048-102
Monoclonal anti-Ig MicroBeads
Anti-MouseIgG1MicroBeads
Positive selection or depletion
130-047-102
Anti-MouseIgG2a+bMicroBeads
Positive selection or depletion
130-047-202
Anti-MouseIgMMicroBeads
Positive selection or depletion
130-047-302
Anti-RatKappaMicroBeads
Positive selection or depletion
130-047-401
Anti-IgGMicroBeads,human
Positive selection or depletion
130-047-501
Product Description Order no.
CD30MicroBeads,human Positive selection 130-051-401
CD30MicroBeads+CD30-PE,human
Positive selection 130-090-505
LineageCellDepletionKit, human
Untouched cell enrichment
130-092-211
Endothelial cells
CD31MicroBeadKit,human
Positive selection or depletion
130-091-935
CD105MicroBeads,human
Positive selection or depletion
130-051-201
CD146MicroBeadKit,human
Positive selection or depletion
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
Product FITC PE APC Biotin VioBlue pure PerCP
Hematopoietic tumor cells
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 - - - -
Product Description Capacity / components Order no.
Hematopoietic tumor cells
MCCD34StemCellCocktail Forcontrolstainingofhumanstemcells 50tests 130-093-427
Product FITC PE APC Biotin VioBlue pure PerCP
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 - - -
Product Description Capacity / components Order no.
Endothelial cells
EPCEnrichmentandEnumerationKit Enumerationofendothelialprogenitorcells 20tests⁶ 130-093-477
ReferencesMore than 13,000 studies used Miltenyi Biotec Products
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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.
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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.
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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.
Order informationPlace your order by fax, phone, or online!
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.
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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.
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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.
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Unlessotherwisespecificallyindicated,MiltenyiBiotecproductsandservicesareforresearchuseonlyandnotfortherapeuticordiagnosticuse.autoMACS,MACS,MACSQuant,CliniMACS,andVioBlueareregisteredtrademarksandgentleMACS,MACSQuantify,MidiMACS,MiniMACS,miRXplore,PIQOR,andµMACSaretrademarksofMiltenyiBiotecGmbH.StemgentandWITareeitherregisteredtrademarksortrademarksofStemgentInc.Copyright©2010MiltenyiBiotecGmbH.Allrightsreserved.
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130-09
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