MACS® Stem Cell ResearchDiscover. Advance. Translate.

Embryonic and induced pluripotent stem cells

Hematopoietic stem cells

Mesenchymal stem cells

Cancer stem cells

Excite and Inspire.Authorized distributor of Stemgent products

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Contents

3 Pioneering research needs pioneering technology

4 Cell separation technology

5 Clinical development

6 Embryonic stem (ES) and induced pluripotent stem (iPS) cells

10 Hematopoietic stem cells (HSCs)

12 Mesenchymal stem cells (MSCs)

14 Cancer stem cells (CSCs)

16 Molecular applications

References: Theproofisinthepublications! Visit www.macs-stemcells.com/references for the extensive list of literature on successful research conducted with our stem cell products.

StemgentproductsarenotdistributedbyMiltenyiBiotecinIsrael,andnotall

StemgentproductsaredistributedintheUS.

Pleasevisitwww.macs-stemcells.comforproduct-specificavailabilityinyourcountry.

For ordering information and the latest news on our products:

www.macs-stemcells.com info@macs-stemcells.com

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Pioneering research needs pioneering technology Complete workflow solutions

Miltenyi Biotec offers a complete portfolio of stem cell products that operates as a comprehensive system for seamless workflow—from sample preparation to downstream applications. Move from stage to stage in your experiment with confidence in a technology that provides for you each step of the way. MACS® Products cover the following fields:

Sample preparation• Convenient,standardized,andtime-savingdissociationofembryoidbodies,

tumors,andothertissueswiththegentleMACS™Dissociator

• Highyields,highcellviability,highreproducibility

• Completedissociationintosingle-cellsuspensionstosupportoptimalresultsduring cell separation or flow cytometry

Cell separation• MACSTechnology:Thegoldstandardincellseparation—proveninover14,500

publications

• Purepopulationsofdifferentadultandpluripotentstemcellsinunderanhour!

• Enrichment/depletionofstemcellprogenyfordifferentiationexperiments

• EasyremovaloffeedercellsfromESandiPScellcultures

• Manualandautomatedcellsorting;scalabletoserveyourneeds

• Applicablefrombasicresearchtoclinicalapplications

Cell analysis• EasilyverifycellseparationresultswithMACSControlCocktails

• Smallfootprintwithbigfeatures:ThecompactMACSQuant®Analyzerfitson yourbenchtopwhilefeaturing9-parameterdetection,absolutecellcounting, and automated compensation

• Arangeoffluorochrome-conjugatesformulti-parameterphenotyping

• Antibodiesandkitsfortheanalysisofvariousstemandprogenitorcells

Cell culture• ExpansionmediaforESandiPScellsandMSCs

• Premium-andGMP-gradecytokinestosupportyourstemcellresearch

• Supplementsforthepreparationofviablesingle-cellsuspensionsfromhuman ESandiPScells

• SpecificallydesigneddifferentiationmediaforCFUassays

• Smallmoleculesforeasymanipulationofstemcellcultures

Molecular applications• Sensitiveone-stepmRNAisolationandcDNAsynthesisformorereliable

quantitativePCRanalysis

• High-qualitymicroarrayservicesforgeneandmicroRNAexpressionprofiling

• SuperAmp™Technologyallowsgeneexpressionprofilingevendowntoafewcells• Straight-forwardisolationofintactmitochondria

MACS®Technology,therecognizedstandardincellseparation,hassupportedresearchforover20years.

Benefit from MACS Technology:• Easilyisolaterarecellpopulations

• Optimalrecoveryandexcellentpurity

• Fast,convenient,andreliable,withnocomplicatedflowsorting needed

• Gentletocells,withflexibilitytoconductseparationinmedia,andlessstressfulcomparedtoflowsorting

• AutomatedcellseparationwiththeautoMACS®Pro Separator

• Compatiblewithflowcytometry

• Cellseparationscaneasilybescaledup

• Abilitytoperformyourexperimentsinasterilesetting

• Bridgesbasicresearchandclinicalapplications

• Havecontroloveryourexperimentswithatechnologythat presents you with ease of use.

Testthetechnologyforyourselfandexperiencehoweasilyitadvancesyourresearch!

About MACS MicroBeads:• Superparamagneticparticles

• Colloidal,foreasyhandlingandshortincubationtimes

• Small(50nm),non-toxic,andbiodegradable:noneedtoremove them from cells after the separation process

• Conjugatedtohighlyspecificmonoclonalantibodiestoensure optimal specificity

TolearnmoreaboutMACSTechnology,including separationstrategiesandthefullrangeofMicroBeads,Columns,andSeparators,visit www.macs-stemcells.com/technology.

Cell separation technology Advance your stem cell research with MACS® Technology

Magnetic labeling

Cellsofinterestarelabeledwith MACS MicroBeads in a short incubationstep.

Magnetic separation

Labeledandunlabeledcells are separated on a MACS Column placed in the magnetic fieldofaMACSSeparator.Theflow-throughcanbecollectedasthenon-magnetic,unlabeledcell fraction.

Elution of the labeled cell fraction

Theseparationcolumnisremoved from the magnetic field and the retained cells are flushed out.

Boththelabeledandunlabeledfractionscanberecovered andusedfordownstreamapp- lications.

MACS Technology

N S

A B C

Figure 1: MicroBead features. Scanning electron microscopy of a cell isolatedwithMACSMicroBeads(A)50nm MicroBeads are so small they canonlybeseenonaTransmissionElectronMicroscope(B).AcrosssectionofaMACSColumnshowingthesteelballmatrix(gray)withthemagneticfieldinwhichlabeledcells(purple)areretained(C).

Figure 2: MACS Cell Separation. CellseparationcanbeperformedmanuallywithaMACSSeparator(A)orautomatedwiththeautoMACSProSeparator(B).

A B

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5

Clinical developmentBench to bedside

Frombenchtobedside.Theprogressofresearchfrombasicsciencethroughpre-clinicalresearchtoclinicalresearchhasseveralcrucialphases(fig.3).

Miltenyi Biotec works with you as a partner to see you through thepivotalprocessesofevolutionfrompre-clinicalresearchintoPhasesI,II,andIIIpriortoattainingapprovalforclinicaluse.Thesephasesarehighlyregulatedandentailadesignfreezeoftheentirestudyatthepre-clinicalresearchstage.

Byusingaportfoliothatmakestransitionsseamless,youcanavoidcostlyandtime-consumingsetbacksinyourwork.

Withourproventrackrecordofover14,500publications,andspecificallyover3,500intheclinicalarena,wegiveyoutheconfidence to move your research forward with products that are designed to comply with each phase of development.

Mindfulofthis,wehavedevelopedanintegratedproductportfolio that provides a smooth transition through each stage ofclinicaldevelopment.Forexample,ourMACS®Cytokineproductsareavailableinresearch,premiumandGMPgrades,ensuringthateachstepoftheprocesscouldn’tbemoreconvenient.

OurexperienceinbringingresearchintotheclinicbeganwhentheCliniMACS®CD34SeparationKitwasCE-marked in1997;morethan10,000patientshavebeentreatedinthelastfive years with cellular products produced using our CliniMACS Technology.

Besidesthepotentialtopropelresearchfrombasicsciencetotheclinic,MiltenyiBiotec’sproductsalsoguaranteethepossibilityforscalabilityinyourresearch,animportantfactorforconsideration.Withourproducts,youcanbesurethattheprotocolsandmethodologyusedinyourresearchcanbesmoothly transitioned to a clinical setting.

When deciding which platform to conduct your experiments on, it is crucial to start and advance your research with a technology you can rely on. Take your research from bench to bedside with no obstacles, using Miltenyi Biotec’s complete product portfolio.

Formoreinformation,pleasevisit www.macs-stemcells.com/clinical.

Figure 3: Thehighlyregulatedphasesofresearchtopre–clinicalresearch,clinicaltrialsandtowardsapplicationsintheclinic.Designfreezesareimplementedatthepre-clinicalstageimplyingasubstantialimpactfinanciallyandtime-wiseifchangesaremadesubsequently.

Highly regulated

ClinicPre-clinical research

Clinical trialsBasic research

Design freeze

Research products Clinical products

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Achieve greater purity, higher yields, and save time compared to traditional cell culture selection methods:

• MACS Technology cuts days and weeks to under an hour

• Sterile separation under your own hood.

ForourfullrangeofESandiPScellseparationproducts,visitwww.macs-stemcells.com/es-ipsc-separate.

Dissociation of embryoid bodies Completedissociationofembryoidbodies(EBs)inESandiPScell differentiation protocols is a prerequisite to allow successful cell separation and analysis. Benefit from Miltenyi Biotec’s innovative gentleMACS™Dissociator,whichhelpsstreamlineandstandardizethisprocessforreliable,reproducibleresults.

• Savetime:effortless,efficient,andreliableautomatedtissuedissociationatthepushofabutton

• Flexible:prepareviablesingle-cellsuspensionsorperformcellhomogenizationfromEBs

• Reducecontamination:closedsystemenablessterilesamplehandling

• Versatile:additionalprogramsandprotocolsfordifferentcellular and molecular applications

Formoredetailsseewww.macs-stemcells.com/gentlemacs.

Cell separation Pure and homogeneous cell populations are essential for investigatingESandiPScellbiology.WithMACSMicroBeadsandKits,youcanisolatespecificcellpopulationswithease:

• Removefeedercellssuchasmouseembryonicfibroblastsforbetterresultsindownstreamapplications(fig.5)

• Depleteunwanteddifferentiatedcellsforuntouchedisolationofyourtargetcellpopulation(fig.6)

• Conductpositiveselectionofhumanormousepluripotentstemcellsusingawidevarietyofmarkers,includingTRA-1-60,CD326(EpCAM),orSSEA-1(fig.7)

ES and iPS cells Sample preparation and cell separation

Figure 5: Efficient depletion of feeder cells. FeederRemovalMicroBeadspermitanefficientdepletionoffeedercellsinlessthan30minutes(A).Puri-tiesofmorethan99%canbeachieved.Incontrast,traditionalmethodsfortheremovaloffeedercellsincludesedimentationandplasticadherence(B)orweaningoffoverseveralpassages(C).Botharetime-consuming, incomplete,andcanresultinasignificantlossofpluripotentstemcells. Fordetailedinformationpleasedownloadposter2from www.macs-stemcells.com/info.

Figure 4: Complete dissociation of EBs with the gentleMACS Disso-ciator. DifferentiationofESandiPScellsfrequentlyinvolvesembryoidbodies(A).Dissociationofthesestructureswithtraditionalmethodsisoftenincompleteandcanyieldhighnumbersofdeadcells(B).gentleMACSTechnologycangenerateviablesingle-cellsuspensionsinastandardized,semi-automatedprocessandtherebyensurereproducibleresults(C). Visit www.macs-stemcells.com/info to download application note 1 and seetheprotocolusedattheNIH.

AB C

Cellharvest,Single-cellsuspension

Mouse/humanESCsor iPSC on feeder cells

Sedimentation/selective adhesion

Weaningoffbyserial passagingon gelatin

3 days45min

B C

3 days45min

Depletionby MACSTechnology usingFeeder RemovalMicroBeads

MACS LS Column 3 min

Magnetic labeling15min

A

Feeder-depletedESCs/ iPSCs

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ES and iPS cells Improved culture and differentiation

Cell differentiation HumanESandiPScellscandifferentiateintovirtuallyanycelltype.MACSTechnologyallowstheseparationofthecellsatdifferenttimepointsduringdifferentiation.Targetcellscanbeisolatedbypositiveselectionifthecellsexpressaspecificsurfacemarkerthatdistinguishesthemfromothercells.HumanCD34,forinstance,wassuccessfullyusedtoisolatehematopoi-eticandendothelialprogenitorsfromdifferentiatedhumanEScellcultures(fig.8).Ifthereisnosuitablemarkerforaparticularcelltype,depletionofunwantedcellsisausefulalternativeforthe isolation of the desired cells.

Mouse Feeder removal

Enrichment of ES and iPS cells

Depletion of pluripotent cells during differentia- tion

Depletion of early differen- tiated cells

FeederRemoval MicroBeads

+

Anti-SSEA-1(CD15) MicroBeads

+1

+

+

Pluripotent Stem Cell IsolationKit

+

Magnetic separation in ES and iPS cell differentia-tion protocols: specifically enrich your target cells• Obtainwell-defined,homogeneouscellpopulations

• Markersavailableformanydifferentiationpathways

• Workwithamorestandardizedprocedurecomparedtoconventional selection in culture

• Noneedfortransgenicselectionmarkers;thuscompatiblewith clinical research requirements

• Separationisfurtherenhancedbypre-dissociationofembryoidbodieswithgentleMACSTechnology

Visit www.macs-stemcells.com/reference which shows publishedexamplesofESoriPScelldifferentiationthatusemagnetic separation.

Figure 6: Ability to remove early differentiated cells from mouse ES cell cultures. PluripotentmouseEScellsshowabroadrangeofSSEA-1expression.Incontrast,only4%ofthesecellsshowexpressionofthenovelmarkerAnti-Diff(A).AculturewhichwasdeprivedofLIFfor2dayswas used to model spontaneous differentiation. In such a culture the expressionprofileofSSEA-1remainsunchanged,whiledifferentiatedcellscanbedetectedeasilywithournewAnti-Diffmarker(B).ThenovelPluripotentStemCellIsolationKit,mouse,isbasedonAnti-Diffandallowsyoutoremoveearlydifferentiatedcells(C).Downloadposter3formoreinformation from www.macs-stemcells.com/info.

Figure 7: Enrichment of pluripotent cells. ESoriPScellculturescancontaindifferentiatedcellsthathavedown-regulatedpluripotencymark-erssuchasCD326(EpCAM)andTRA-1-60(A).CellsisolatedbymagneticseparationusingthePluripotentStemCellMicroBeadsortheAnti-TRA-1-60MicroBeadKitdonotshowunwanteddifferentiation(B)andmaintainnormalkaryotypesformorethan6months(C).

Figure 8: Enrichment of target cells in ES or iPS cell differentiation protocols. HumanEScellswereculturedintheabsenceofgrowthfactorstoinducedifferentiation(A).After10days,5–10%ofthecultureconsistedofCD34-positiveprogenitors(B).MACSTechnologyandCD34MicroBeadsallowedrapidisolationofthiscellpopulationwithhighpurity(C). Upto108cellswereusedperseparation.Fordetailedinformationvisit www.macs-stemcells.com/infoanddownloadcustomerreport4byWanget al.

Table 1:ExamplesofmagneticseparationinmouseESandiPScellresearch.

1PositiveselectionofESandiPScells.

SSC-H

95%

CD34

95%<0.1%

SSC

CD34-APC

5.2%

SSC

CD34-APC

A B C

CD34-APC CD34-APC CD34-APC

SSC

SSC

SSC

BA

Anti-Ha-1-60-PE

CD

326-

AP

C

TRA-1-60-PE

CD

326

-AP

C

Anti-Ha-1-60-PE

CD

326-

AP

C

TRA-1-60-PE

CD

326

-AP

C

C

A B C

Anti-Diff-PEAnti-Diff-PE Anti-Diff-PE

An

ti-S

SEA

-1-A

PC

An

ti-S

SEA

-1-A

PC

An

ti-S

SEA

-1-A

PC

10³-1-101

10¹ 10²0

10³

10²

10¹

1

Anti-Diff-PE

Anti-SSE

A-1-APC

35%

10³-1-101

10¹ 10²0

10³

10²

10¹

1

Anti-Diff-PE

Anti-SSE

A-1-APC

2%4 %

10³-1-101

10¹ 10²0

10³

10²

10¹

1

Anti-Diff-PE

Anti-SSE

A-1-APC

95%5.2%<0.2%

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ES and iPS cells Optimal cell analysis

Figure 9: Easy histochemical staining. Alkalinephosphatase(AP)isex- pressedatelevatedlevelsinundifferentiatedpluripotentcells(A).Stemgent’s APStainingKitIIprovidesaneasyimmunohistochemicalstainingprotocoltoassessexpressionofthisphenotypicmarker.DetectionofadditionalstemcellsurfacemarkerssuchasCD324(B),TRA-1-60(C)andTRA-1-81complementsthephenotypicanalysis.Thiscanbedonebyimmunochem-istryorevendirectlyinculturewithStemgent’sStainAliveantibodies(D).

• UseouruniqueAnti-Feederantibodiestoexcludefeedercells from your flow cytometric analysis

• Best-in-class:theMACSQuantAnalyzerisabenchtopflowcytometerwithabsolutecellcountingcapability

ForourfullrangeofESandiPScellanalysisproducts,visit www.macs-stemcells.com/es-ipsc-analysis.

• Pre-defined sets of established pluripotency markers

• Live cell staining with Stemgent’s StainAlive antibodies

• Analysis of surface markers and transcription factors

Cell analysis MACSAntibodiesincombinationwiththeMACSQuantAnalyzeroffer you great solutions for cell analysis. We can provide you withevenmoresolutionsforstemcellanalysis,includingStemgent’sproducts.Thecombinedportfoliobringsyouabroadrangeofhigh-qualityantibodies,ensuringallyourneedsare conveniently met.

• Easeofuse:Evaluatethedifferentiationstatusofhuman andmouseESandiPScellswitheaseusingthedetectionkitforalkalinephosphataseactivity,aphenotypicmarkerofpluripotent stem cells

• Convenient:Livecellstainingantibodiesenablereal-timeimmunocytochemistryofviablecells—nofixationrequired;continuedculturepossible

• Comprehensiverangeoffluorochromeconjugatesavailable(VioBlue®,PerCP,PE,APC,FITC,DyLight™,StainAlive®)tomeet even multiparameter imaging requirements

Human Feeder removal

Enrichment of iPS cells after repro-gramming

Enrichment of pluripotent cells during culture

Enrich- ment of cardiovas- cular pro- genitors

FeederRemovalMicroBeads,mouse

+2

Anti-FibroblastMicroBeads,human

+3

Anti- TRA-1-60 MicroBeadKit

+1

+

+

Pluripotent Stem Cell MicroBeads,human

+1

+

+

Anti-SSEA-1(CD15)MicroBeads

+4

1PositiveselectionofESandiPScells 2 If using mouse feeder cells 3 If using human feeder cells 4HumanpluripotentcellsareCD15-negativeandtherebyremoved inthisstep.Generalprotocolsforcompletedepletionofpluripotentcells are under development.

Table 2: ExamplesofmagneticseparationinhumanESandiPSresearch.

CD324 (E-Cadherin)-APC

Rel

ativ

e ce

ll n

um

ber

CBA D

Marker Mouse ES and iPS cells

Human ES and iPS cells

Alkaline phosphatase + +

CD324 – +

CD326 – +

Nanog + +

Oct4 + +

Sox2 + +

SSEA-1 + +

SSEA-3 – +

SSEA-4 – +

TRA-1-60 – +

TRA-1-81 – +

Rex1 + +

c-Myc – +

Klf4 + +

Table 3: MarkersformouseandhumanESandiPScellresearch.

Rel

ativ

e ce

ll n

um

ber

CD324 (E-Cadherin)-APC

9

Stemgent, mouse primary iPS and human fibroblast cell lines enhance your reprogramming and differentiation studies in basic and translational research. Stemgent cell lines closely mimic the in vivo state and generate more physiologically relevant data.

ForourfullrangeofESandiPScellcultureproductsmentioned,visit www.macs-stemcells.com/es-ipsc-culture.

Figure 11: hES Cell Cloning and Recovery Supplement—clearly advan-tageous. HumanH1EScellsweretrypsinizedandsingle-cellsuspensionswerereseededatdifferentcellnumbersasindicatedinNutriStemXF/FFCultureMedium(A).AdditionofthehESCellCloningandRecovery SupplementimprovessurvivalofsolitaryESandiPScellsover30-fold(B)andisalsoadvantageousforallapplicationsinvolvingsingle-cell suspensions,suchasseparationsbyMACSTechnologyandcellanalysis.

ES and iPS cells Guaranteed cell culture

Cell culture TherightmediumisvitalforensuringESandiPScellculture.OurcomprehensiveselectionincombinationwithStemgent’sportfolio provides you with:

• Ready-to-usemediaandcellculturesupplementsthatguaranteeyouoptimalandstandardizedcultureconditionsduringexpansionandmaintenanceofpluripotentESand iPS cell lines

• Abroadrangeofrecombinantcytokinesandgrowthfactors(e.g.FGF-2,EGF,SHH,LIF,Wnt-3a,Wnt-5a,ActivinA)tosupplement your media according to your needs and individual differentiation protocols

• AwaytotakeyourresearchfrombenchtobedsidewithourcytokinesavailablefromresearchtoGMPgrades

• AuniqueportfolioofStemgentsmallmolecules,potenttoolsthatallowyoutomanipulatecellreprogramming,self-renewal,anddifferentiation

NutriStem™ XF/FF Culture Medium

• Xeno- and feeder-free culture (fig. 10)

• Low amounts of FGF-2 and TGF-β closer to physiological levels

• Ideal for human iPS cell generation

hES Cell Cloning & Recovery Supplement (Thiazovivin)

• Improves survival of single human ES cells by more than 30-fold after thawing, dissociation, and replating (fig. 11)

• Crucial for preparing viable single-cell suspensions for flow cytometry and cell separation

Small molecules with results you want:

• Easy cell penetration: simply add them to your culture media

• Low antigenicity

• Tested for cytotoxicity on stem cells • Download poster 5 at www.macs-stemcells.com/info

to learn how small molecules act

Save time and effort with Stemgent’s ready-to-use retroviruses for cell reprogramming.

B

A

Figure 10: NutriStem XF/FF Culture Medium: improved human ES and iPS cell culture. ColoniesofthehumanEScelllineH1culturedin NutriStemXF/FFCultureMediumshowtypicalEScellmorphologyandmaintainpluripotencyandnormalkaryotypesoverlong-termculture.

2,500 cells 10,000 cells 40,000 cells

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Cell separation HSCresearchhascontributedagreatdealtounderstandinghematopoiesis for use within clinical settings. Conduct your experimentsinastandardized,seamlessprocesswithproductsoptimizedfordirectusewitheachother:

• DirectlyisolateHSCsorhematopoieticprogenitorcells(HPCs)witharangeofdifferentmarkers(table4);fastandeasy,fromanysource

• EnrichuntouchedHSCsandHPCsusingourLineageCellDepletionKits,mouseorhuman

• Performsequentialsortingtodefinesubpopulationsbasedonseveralmarkers(fig.13)

• Easilyobtaincommittedcellsforcomparisonusingrelatedseparation products from our portfolio of mature lineage markers

ForourfullrangeofHSCseparationproducts,visit www.macs-stemcells.com/hsc-separate.

• Decrease processing time and stress to cells with magnetic separation prior to flow sorting

• Take advantage of CD133, a unique marker for primitive HSCs

• Available for use in clinical settings: CD34 and CD133 MicroBeads as CE-marked reagents, manufactured under GMP conditions *

• Proven technology: CD34 MicroBeads have been cited more than 2,000 times since their launch in 1993

Marker/ MicroBead Kit

Mouse Human

CD341 +

CD1051 +

CD117 + +

CD133 +

Sca-1 +

Linneg + +

Linneg/CD34+ +

Linneg/CD133+ +1alsoavailablewithMultiSortoption

Table 4: MarkersfortheisolationofHSCsandHPCs.

HSCs Cell separation— trusted in research and the clinic for more than 15 years

Figure 12: Save time on HSC isolation. HSCsrepresentaminorfractionofhumanperipheralbloodmononuclearcells(PBMCs),identifiedhereasCD34+CD45lo(A)orCD34+CD133+cells(C).Whetherintheresearchorclinicalsetting,MACSTechnologyprovidesahighlyefficientwayofisolatingpurepopulationsofthisrarefractionusingeitherCD34MicroBeads(B)ortheCD133MicroBeadKit(D).

Figure 13: Efficiently enrich mouse progenitor cells. MouseHSCsandearlyHPCsarecontainedintheLinneg CD117hicompartment(A).Usingthe LineageCellDepletionKit,mouse,followedbypositiveselectionwith CD117MicroBeads,HSCsandHPCscanbeefficientlyenriched(B).

A B

CD117-PE

Lin

eag

e m

arke

rs (C

D5,

CD

11b

, C

D45

R, a

nti

-Ly-

6g, 7

-4,

Ter-

119)

-Bio

tin

/An

ti-B

ioti

n-A

PC

CD117-PE

Lin

eag

e m

arke

rs (C

D5,

CD

11b

, C

D45

R, a

nti

-Ly-

6g, 7

-4,

Ter-

119)

-Bio

tin

/An

ti-B

ioti

n-A

PC

CD117-PE CD117-PE

Lin

neg

-AP

C

Lin

neg

-AP

C

D

CD133/2-PE

CD

34-F

ITC

C

CD133/2-PE-A

CD

34-F

ITC

CD33/2-PE

CD

34-F

ITC

B

CD34-FITC

CD

45-A

PC

CD34-FITC

A

CD

45-A

PC

CD34-FITC

CD

45-A

PC

CD34-FITC

CD

45-A

PC

CD33/2-PE

CD

34-F

ITC

*Foravailabilityinyourcountry,pleasecontactyourlocalsalesrepresentative.

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HSCs Comprehensive cell culture and analysis

Cell analysis InHSCresearch,characterizingthesurfacephenotypeof yourtargetcellsiscriticaltoascertainthecellularsubsetyou areworkingwith.Utilizeourwiderangeofproductsforflow cytometry or microscopic investigations to make the phenotyping process easier:

• Profitfromourcollectionoffluorochrome-conjugatedhematopoieticmarkerstoanalyzestemcellaswellaslineagemarkersforyourcellsbyflowcytometry

• MACSAntibodiesareperfectlysuitedtoourseparationreagents.Getreproduciblestainingresultsandavoidinaccessibleepitopeswithourrecommendedstainingprotocols

• OurMACSControlCocktails,availableforCD34andCD133,allow easy and convenient analysis of your separation results(fig.14)

TheentirerangeofHSCcellanalysisproductsisavailableatwww.macs-stemcells.com/hsc-analysis.

Endothelial progenitor cells (EPCs) are part of the CD34+ population. The EPC Enrichment and Enumeration Kit utilizes EPC markers for efficient magnetic pre-enrichment by MACS Technology and subsequent sensitive cell enumeration by flow cytometry. Visit www.macs-stemcells.com/info for poster 6 with further information.

Figure 14: Convenient evaluation of separation results. TheMCCD34/CD133StemCellCocktailwasusedtoevaluateseparationresultsobtainedwithCD133MicroBeads.GatingonviableCD45+CD34+cells,primitiveCD133+ HSCscanbereadilyenumeratedinpre(A)-andpost(B)-separationsamples.Usingthe‘Easymode’functionforautomatedgatingontheMACSQuantAnalyzermakestheprotocolevenmoreconvenient.

CD133/2-PE

CD

34-A

PC

P1/P2/P3/P4

B

CD133/2-PE

CD

34-A

PC

A

CD133/2-PE

CD

34-A

PC

P1/P2/P3/P4

CD133/2-PE

CD

34-A

PC

Cell culture StimulateandcultureyourHSCswiththeMACSCellCultureportfolio:

• MACSHSC-CFUMedia,evaluatethenumberofHSCsinyourstarting material and assess their differentiation potential as colony-formingunits.Seeposter7at www.macs-stemcells.com/info for more information on the HSC-CFUmedia

• MACSCytokinesarereliablesupplementsforroutinecultureas well as delicate differentiation experiments. Benefit from lowendotoxinlevels,highpurity,andtestedbiologicalactivity,andsupplementyourbasicmediaaccordingtoyourneeds

ForourfullrangeofHSCcellcultureproducts,visit www.macs-stemcells.com/hsc-culture.

Take your research from bench to bedside with MACS Cytokines, available in research, premium, and GMP grades. Commonly used cytokines in HSC research include Stem Cell Factor (SCF), FLT3-Ligand, and Thrombopoietin (TPO).

Figure 15: Reliable evaluation of human HSC differentiation potential. Schematicdiagramshowingcolony-formingunits(CFUs)formedby multipotentandlineage-committedhematopoieticprogenitors.

CFU-M

CFU-GM

CFU-G

BFU-E

CFU-E

CFU-GEMM

12

MSCs Standardize your cell expansion and differentiation potential

Cell separation Work with purified cell populations to fully explore the potentialofMSCs,whichholdgreatpromiseforresearchandtherapeutic applications.

• EasilyisolateMSCsfromhumanbonemarrow,lipoaspirate, orothertissuesusingawiderangeofmarkers(table5)

• ObtainMSCsdirectlyfromtissuefordownstream experiments

• Depletecontaminatingcellsfromyourhumanormouse MSC cultures

• EnrichmousebonemarrowMSCsafterexpansioninculture

• Usetheready-to-goresearchtoolboxestoisolateandexpand your human MSCs

“… Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multi-potent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. …” Selim Kuçi et al. Visit www.macs-stemcells.com/references for the full reference.

Understanding mouse MSCs is an ongoing effort. We offer a wide range of MicroBeads and fluorochromes for you to choose from for your mouse MSC research needs.

Findoutmoreatwww.macs-stemcells.com/msc-separate.

Marker Source tissue

CD271 Bonemarrow,lipoaspirate

MSCA-1 Bone marrow

Anti-fibroblastantigen Bone marrow

CD105 Bone marrow

CD146 Umbilicalcordblood,lipoaspirate, dentalpulp,endometrialtissue

Table 5: MACSTechnologyisolatesdefinedhumanMSC populationswithhigh-purity.Specificmarkersforspecificsourcetissues.

Figure 17: Isolate highly purified MSCs in under an hour. HumanbonemarrowMSCsalsoexpressthenovelsurfacemarkerMSCA-1.MSCA-1+ cells areshowntobeCD45–andCD271+.UsingMSCA-1MicroBeads,highly purifiedMSCscanbeisolatedfrombonemarrowinunderanhour.

Anti-MSCA-1 (W8B2)-APC

CD

45-F

ITC

Anti-MSCA-1 (W8B2)-APC

CD

45-F

ITC

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Figure 16: Clinical-scale isolation of CD271+ MSCs.Inhumanbonemarrow,MSCsarecontainedintheCD271+compartment(A),whichcanbeefficientlyenrichedusingtheCD271MicroBeadKit(B).Furthercharacteriza-tionrevealsthatMSCswithCFU-FdifferentiationpotentialareexclusivelypresentintheCD271+fraction(C)andhaveahigherclonogenicpotentialthancellsisolatedbyconventionalplasticadherence(PA)(D).Tolearnmoreaboutclinical-scaleisolationofCD271+ MSCs download poster 8 at www.macs-stemcells.com/info.

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Cell analysis IdentifyandenumerateMSCsfrombonemarrow,lipoaspirateandothertissuesbyflowcytometryorfluorescencemicroscopywithourrangeofantibodies.

EnsureaccurateandreliablephenotypingofMSCswiththeMSCPhenotypingKit,aready-to-usecocktailcontainingallsixmarkersforMSCcharacterization,recommendedbytheISCT orwithourantibodiesforindividualmarkers.

ForourfullrangeofMSCcellanalysisproducts,visit www.macs-stemcells.com/msc-analysis.

MSCs Improve cell analysis and culture

Cell culture ExpansionofisolatedMSCsandevaluationoftheirdifferentiationpotentialarecriticalstepsinbasicMSCresearchandapre-requisite for clinical applications.

• MACSNHExpansionMediaareoptimizedtoprovidethemost convenient expansion of MSCs from various sources. CombinationwithourseparationproductsforMSCisolationguarantees high expansion rates

• MACSNHCFU-FMediumsupportsyouinenumeratingthenumberofMSCsinyoursample

• MACSNHDifferentiationMediaallowyoutoexplorethefulldifferentiationcapacityofyourcells,beitforadipocytic,chondrocytic,orosteoblasticlineages

• MACSCytokinesareavailableinresearch,premiumandGMPgradestotakeyoufrombenchtobedside.CommonlyusedcytokinesinMSCresearchincludeFGF-1,FGF-2,EGF,TGF-β,andBMP-2

ForourfullrangeofMSCcellcultureproducts,visit www.macs-stemcells.com/msc-culture.

CytoMix-MSC, human is a composition of cytokines for the most efficient and reproducible expansion of human MSCs, resulting in 50% more cumulative population doublings. Full results are in poster 9 at www.macs-stemcells.com/info.

Figure 18: ISCT guidelines–compliant MSC analysis. MSCs isolated with theCD271MicroBeadKitwereexpandedincultureandanalyzedforthe expressionofsurfacemarkers.TheMSCPhenotypingKitincombinationwith theMACSQuantAnalyzerguaranteesrobuststainingforISCT-standardizedmarkers and straightforward analysis.

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Figure 19: Reliable expansion and differentiation of MSCs. In MSC research,expansionanddifferentiationofMSCsareroutinelyperformed.ExpandMSCsinlesstimewiththeNHExpansionMediaandCytoMix-MSC,humancytokinecocktail.MSCscanbedifferentiatedintoadipocytes, chondrocytes,orosteoblastsusingourspecialdifferentiationmedia.

AdipocytesNHAdipoDiffMedium

ChondrocytesNHChondroDiff Medium

OsteoblastsNHOsteoDiffMedium

MSC expansionNHExpansionMedium

MSC enumerationNHCFU-FMedium

NH stem cell source, e.g., bone marrow, lipoaspirate

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Cell separation Understandingtheroleofdifferenttumorcelltypesisthekeytodiscoveringfuturetherapies.Tumorsareaheterogeneousmixofmultiplecelltypes.IsolateCSCswithMACSTechnologytoobtainhighpuritiesandyieldsofyourtargetcells.

• Widerangeofreagentsformanymarkerscommonlyexpressedontumorsamples(table6)

• Useindirectmagneticlabelingforcustomizedcell separations:anycell,anyspecies,anyprimaryantibody

• Isolatecellsonyourownschedulewheneveryoursample isready(fig.21)

ForourfullrangeofCSCseparationproducts,visit www.macs-stemcells.com/csc-separate.

Figure 20: Efficient tumor dissociation. HumanmelanomametastasesweredissociatedusingthegentleMACSDissociatortumordissociation program.Afterdissociation,thesingle-cellsuspensionsarecomposedmainlyoftumorcells(TC),tumor-infiltratinglymphocytes(TIL),and erythrocytes(ERY).Downloadposter10formoreinformationat www.macs-stemcells.com/info.

TIL

ERY

TC

CSCs Whenever you need it—from tumor tissue to purified CSCs

Tumor type Cell surface marker

Acutemyeloidleukemia(AML) CD34+/CD38–

Breast cancer EpCAM (ESA)+/CD44+/CD24–/Lineage–

Ovariancancer CD133+

CD44+/CD117+

CD24+

Glioblastoma CD133+

CD15+

Medulloblastoma CD133+

CD15+

Smallcellandnon-small cell lung cancer

CD133+

Hepatocellularcarcinoma CD45–/CD90+

Prostate cancer CD44+/α₂β₁hi/CD133+

Colon cancer CD133+

CD44+

CD26+

Melanoma CD20+

ABCB5+

CD271+

Pancreas adenocarcinoma CD44+/CD24+/EpCAM (ESA)+

Renalcarcinoma CD133enhancesvascularization

Headandnecksquamouscellcarcinoma(HNSCC)

CD44+

Table 6: Cell surface markers of CSCs in different types of tumors. .MiltenyiBiotecoffersabroadportfolioofantibodiesfortheanalysisoftumorsamplesandtheisolationofCSCs.Forthecompleteportfolio,pleasevisit www.macs-stemcells.com/csc.

Sample preparation Breakingupsolidtumortissueintoviablesingle-cell suspensionsisachallenge.Consistent,reliabledissociationiseven more imperative when patient material is limited and precious.ThegentleMACSDissociatorstreamlinesandstandardizessamplepreparationusingacombinedmechanicalandenzymaticprocessforreliableandreproducibleresults.

• Specializedprograms,protocols,andkitsthatareoptimizedfor tumor dissociation

• Efficientgenerationofsingle-cellsuspensionsofviablecells

• User-independentmechanicalprocessingensureshighreproducibility

• Efficient,automatedtissuedissociationatthepush ofabutton

• Closedsystemthatguaranteessterilesamplehandling

• Optimalpreparationoftissueforsubsequentflow cytometric analysis or magnetic separation

Formoredetailsseewww.macs-stemcells.com/gentlemacs.

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Cell analysis DetectandanalyzeCSCsandothertumorcellswithour antibodies:

• Largeselectionofantibodiesforthephenotypic characterizationofvariouscelltypes

• Varietyofdyeconjugatesforeffectivemultiparameter flow cytometry or sophisticated multi spectral imaging experiments

CSCs are characterized by different markers, depending on the tissue source (table 6). Regardless of the type of tissue you work with, we provide antibodies for your analysis.

ForourfullrangeofCSCanalysisproducts,visit www.macs-stemcells.com/csc-analysis.

CSCsExtensive markers for cell analysis

Figure 21: Convenient isolation of CSCs. CSCs can express different mark-ersdependingonthetissuesourceorcellline,e.g.CD44+(A)orCD24–/CD44+ (B).IsolatedCD44+orCD24–/CD44+cellswereobtainedusingeitherCD44MicroBeadsalone(C)orincombinationwiththeCD24MicroBeadKit(D),respectively.MicroBeadscanbeusedtoisolateCSCswithhighpurityfromcancer cell lines or primary tissue.

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Takeyourstemcellresearchandanalysistothesubcellularandmolecular level.

One-step mRNA isolation and in-column cDNA synthesisPremiummRNAisisolatedwithin15minutesdirectlyfromcellsortissues.TheμMACS™One-stepcDNAKitcombinesefficientmagneticisolationofmRNAwithrevolutionaryin-columncDNAsynthesis.PurifiedcDNAcanbegeneratedfromjustafewtoasmanyas107cells(fig.23).

ChIP-in-a-column with MACS TechnologyTheμMACSProteinA/GMicroBeadsimprovestandardimmunoprecipitation and significantly accelerate the search for interactingproteins.Chromatinimmunoprecipitation(ChIP)protocolsalsobenefitfromthehigherspecificityandlowerbackgroundbindingofμMACSProteinA/GMicroBeads.

Isolation of human mitochondriaAchieve higher yields of functional mitochondria as compared todifferential–andultracentrifugationprotocols.Thekit’sprocedurereliesontherenownedMACSTechnology.Aftercelllysis,anoutermitochondrialmembranemoleculeisusedtomagnetically isolate functional organelles.

Enhanced transfection of stem cellsStemfect™TransfectionReagentsarepolymer-basedandspecifically designed for in vitroDNAtransfectionofstemcells.Thesetransfectionreagentsensurehightransfectionefficiencies and low cytotoxicity.

Enrichment of transfected cellsEnrichmentoftransfectedcellswithMACSelect™ Systems takes 3hoursversusseveralweeksforantibiotictreatment.

Molecular applicationsKits to advance your stem cell research

Figure 23: Principle of MACS Technology for mRNA isolation and cDNA synthesis.SensitivemagneticisolationofmRNAcanbeperformedwithinminutes.Subsequentin-columncDNAsynthesispreventsthelossofmaterialtofurtherincreasesensitivityandreliability.

Cells or tissue are lysed using Lysis/BindingBuffer.

Clearing of lysate using LysateClear Column.

AdditionofµMACS™Oligo(dT)MicroBeads to the lysate.

5 min

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Lysate is applied to a µColumninthermoMACS™Separator.WashofmRNA.

0 min

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Purification and release of cDNA/mRNAhybrid.

1 h

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Figure 24: Overview of genomic services workflow.

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Ten years of microarray experienceMiltenyi Biotec has ten years of microarray experience and offersahugevarietyofgenomicservices.Since2003,wearealso an officially certified Agilent Service provider.

Thereisnoneedtoestablishmicroarraytechnologyinyourownlaboratory—simplysendcells,tissue,orbloodsamplestoourMicroarrayServicesDepartmentandreceivereliableresultsand detailed documentation in return.

Flexible expression profiling servicesMiltenyiBiotecprovidesawiderangeofcost-effectivemicroarray services:

• microRNAexpressionanalysisbasedon miRXplore™Microarrays

• Agilentwholegenomeexpressionanalysis

• Agilentarray-CGH

• BioinformaticsServices

SuperAmp™ Service for one-cell microarray experimentsTheSuperAmpServiceisanextensionoftheMicroarrayServices and allows successful gene expression profiling from 1–10,000cells.

Visit www.macs-stemcells.com/molecular to see the whole molecular applications portfolio.

Molecular applicationsFirst class genomic services for your stem cell research

Send sample — receive results

Technical support for experimentaldesign and microarray selection

• microRNAmiRXplore™Microarrays

• AgilentWholeGenomeMicroarrays

RNA extraction and quality control

Optional:

• Amplificationandqualitycontrol

• SuperAmpService*:1–10,000cells

Synthesis and purification of fluorescently labeled probes

Microarray hybridization

Image capture and analysis of primary data

Optional: Bioinformatics Services**

• Pre-processing

• RatioBuilding

• Cluster

• DiscriminatoryGenes

• FunctionalGrouping

• PathwayAnalysis

Results and report

• DataonCD-ROM

*microRNAscannotbeamplifiedwiththeSuperAmpService.**PleaseinquireformicroRNABioinformaticsServices.

Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/localtofindyournearestMiltenyiBioteccontact.

MACS,gentleMACS,autoMACS,CliniMACS,MACSQuant,MACSlogo,MACSelect,VioBlue,µMACS,miRXplore,andSuperAmpareeitherregisteredtrademarksortrademarksofMiltenyiBiotecGmbH.StainAlive,Stemgent,Stemfect,andNutriStemareeitherregisteredtrademarksortrademarksofStemgentInc.Allothertrademarksmentionedinthisdocumentarethepropertyoftheirrespectiveownerandusedforidentificationpurposesonly.Unlessotherwisespecificallyindicated,MiltenyiBiotecproductsandservicesareforresearchuseonlyandnotfortherapeuticordiagnosticuse.Copyright©2011MiltenyiBiotecGmbH.Allrightsreserved.

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www.miltenyibiotec.com

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