macs stem cell quick guide2013

Mesenchymal stem cellsDriving your MSC research

Human samples Mouse samples

Phenotyping of expanded MSCs

Human• MSCPhenotypingKit• Fluorochrome-conjugatedantibodiesagainst,e.g.,CD73,CD90,CD105,CD45,CD34,CD14,CD20,etc.

Mouse• Fluorochrome-conjugatedantibodiesagainst,e.g.,Lineagemarkers,CD105,CD117,Anti-Sca-1,CD45,CD116,etc.

• MACSQuant®FlowCytometers

Molecular analysis of MSCs

• mRNAIsolationKitandcDNASynthesisKit(forRT-PCR)• ProteinIsolationKits(forChIPornormalIPs)• Microarrayservices• SuperAmp™Technology• Bioinformatics• MultiMACS™M96Separator• Mitochondriaisolation

Differentiation of MSCs

Human• NHDifferentiationMedia• Cytokines,e.g.,TGF-β• NHCFU-F

Mouse• Cytokines,e.g.,FGF-8b

Characterization of MSCs

Sample type

Frozen• DeadCellRemovalKit• Pre-SeparationFilters

Tissues• gentleMACS™Dissociator• Pre-SeparationFilters

Phenotyping of MSCs prior to expansion

Fluorochrome-conjugated antibodies against, e.g.,• CD271• Anti-MSCA-1

• CD146• CD105• CD45

Fluorochrome-conjugated antibodies against, e.g.,• CD45

• Anti-Ter-119• Anti-Sca-1• CD105

Isolation of MSCs

Magnetic isolation• CD271(LNGFR)MicroBeadKits• Anti-MSCA-1(W8B2)MicroBeadKit

• CD90MicroBeads• CD105MicroBeads• CD146MicroBeadKit• CD73Antibodies• LineageCellDepletionKit

Flow sorting• Fluorochrome-conjugatedantibodies(markersaslisted)

Plastic adherence• NHExpansionMedia• CytoMix–MSC

Expansion of MSCs

• NHExpansionMedia

• CytoMix–MSC

• Cytokines,e.g.,FGF-2,PDGF-BB,TGF-β

Isolation of MSCs

Magnetic isolation• LineageCellDepletionKit• CD105MultiSortKit(PE),CD117MicroBeads,Anti-Sca-1MicroBeadKit(FITC)

• CD45MicroBeads,Anti-Ter-119MicroBeadsremovalpre-culture

• CD11bMicroBeadsremovalpost-culture

Plastic adherence

Expansion of MSCs

• Cytokines,e.g.,TGF-β,EGF

Translate research to clinical applications• GMPCytokines• CliniMACS®System• CliniMACSReagents,e.g.,CD271

Samplepreparation Cellseparation Cellanalysis Cellculture Molecularapplications Clinicalapplications

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Sample type

• CD146• CD105• CD45

Isolation of MSCs

Expansion of MSCs

• CytoMix–MSC

Isolation of MSCs

Plastic adherence

Expansion of MSCs

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Samplepreparation Cellseparation Cellanalysis Cellculture Molecularapplications Clinicalapplications Samplepreparation Cellseparation Cellanalysis Cellculture Molecularapplications Reprogramming

Embryonic and induced pluripotent stem cellsPioneering solutions for ES and iPS cells research

Hematopoietic stem cellsMaking headway in your HSC research

iPS cells

Do you differentiate your ES or iPS cells?

Characterization of reprogrammed cells

Human• ExtracellularPSCstaining:TRA-1-60,TRA-1-81,SSEA-3,SSEA-4,CD326,CD324,etc.

• Extracellulardifferentiatedcellstaining:SSEA-1

• Transcriptionfactorstaining:Oct4,Sox2,Nanog,etc.

Mouse• ExtracellularPSCstaining:SSEA-1• Extracellulardifferentiatedcellstaining:Anti-Feederantibodies

• MACSQuantFlowCytometers

Isolation of reprogrammed cells

Magnetic isolationHuman• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads• Anti-SSEA-4MicroBeads• IsolationofTRA-1-81+cellsviaindirectmagneticlabeling

• hESCloningandRecoverySupplement

Mouse• Anti-SSEA-1MicroBeads

Flow sorting• Fluorochrome-conjugatedantibodies(markersabove)

Colony Picking• StainAlive™Antibodies

Generation of iPS cells

Human• mRNAReprogrammingReagents• Pluriton™ReprogrammingMedia• Smallmolecules

Mouse• ReprogrammingEcotropicRetrovirus• SmallMolecules

Differentiation of ES or iPS cells

Human or mouse• Cytokines• Smallmolecules

Note:Differentcytokinesareusedinaccordancewiththedifferentiationpathway.

Purification of ES or iPS cells prior to differentiation

Human• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads

Mouse• PluripotentStemCellIsolationKit

Differentiation of ES or iPS cells via embryoid bodies

• EmbryoidBodyDissociationKit• gentleMACS™Dissociator

Monitoring differentiation by flow cytometry or immunohistochemistry

• Antibodies,forexample,useCD34antibodiestoanalyzecellsduringdifferentiationintohematopoieticorendotheliallineages.

Purification of cells during or after differentiation

MicroBeadreagents,forexample,cellseparationreagentsfordifferentiatedcells,e.g.,• NeuralCrestStemCellMicroBeads• CD34MicroBeads• CD144(VE-Cadherin)MicroBeads• CD309(Flk-1)MicroBeadKit• Anti-PSA-NCAMMicroBeadKit• Anti-A2B5MicroBeadKit

Sample type

PBMNCs or lysed whole blood• RedBloodCellLysisSolution

Isolation of HSCs

Magnetic isolation• LineageCellDepletionKit +CD117MicroBeads +Anti-Sca-1MicroBeadKit

(FITC)• CD105MultisortKit(PE)+Anti-Sca-1MicroBeadKit(FITC)

Flow sortingFluorochrome-conjugatedantibodiesagainst,e.g.,• Lineagemarkers• Anti-Sca-1• CD117(c-kit)• CD135(Flt3R)Isolation of HSCs

Magnetic isolation• CD34MicroBeadKit• CD133MicroBeadKit• LineageCellDepletionKit• CD117MicroBeadKit

Flow sortingFluorochrome-conjugatedantibodiesagainst,e.g.,• CD34• CD133• CD45• CD90• CD38

Expansion of HSCs

• Cytokines,e.g.,TPO,SCF,Flt3

Expansion of HSCs

• Cytokines,e.g.,SCF,Flt3

Phenotyping of HSCs

HumanFluorochrome-conjugatedantibodiesagainst,e.g.,• CD34• CD133• CD45• CD90• CD38• MACSQuant®FlowCytometers

MouseFluorochrome-conjugatedantibodiesagainst,e.g.,• Lineagemarkers• Sca-1• CD117(c-kit)• CD135(Flt3R)• MACSQuant FlowCytometers

Molecular analysis of HSCs

• mRNAIsolationKitandcDNASynthesisKit(forRT-PCR)

• ProteinIsolationKits(forChIPornormalIPs)

• Microarrayservices• SuperAmp™Technology• Bioinformatics• MultiMACS™M96Separator• Mitochondriaisolation

CFU assays for HSCs

Human• HSC-CFUMedia

Differentiation of HSCs

Human or mouse• Cytokines

Characterization of HSCs

ES cells

Transfection of ES or iPS cells

Human• Stemfect™hESCDNATransfectionKit• StemfectEnhancedhESCDNATransfectionKit• StemfectRNATransfectionKit• MACSelect™Products

Mouse• Stemfect2.0-mESCDNATransfectionReagent• MACSelect™Products

Single-cell passaging of ES or iPS cells

Human• hESCloningandRecoverySupplement• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads

Monitoring of pluripotency by flow cytometry or immunohistochemistry

Human• ExtracellularPSCstaining:TRA-1-60,TRA-1-81,SSEA-3,SSEA-4,CD326,CD324,etc.• Extracellulardifferentiatedcellstaining:SSEA-1• Transcriptionfactorstaining:Oct4,Sox2,Nanog,etc.• MACSQuant®FlowCytometers

Mouse• ExtracellularPSCstaining:SSEA-1• Extracellulardifferentiatedcellstaining:Anti-Feederantibodies• Transcriptionfactorstaining:Oct4,Sox2,Nanog,etc.• MACSQuant®FlowCytometers

Translate research to clinical applications• GMPCytokines• CliniMACSSystem• CliniMACS®Reagents,e.g.,CD34,CD133

Removal of feeder cells

Depleting feeder cellsHuman• Anti-FibroblastMicroBeads

Mouse• FeederRemovalMicroBeads

Isolating pluripotent stem cellsHuman• Anti-TRA-1-60MicroBeadKit

• PluripotentStemCellMicroBeads

Culture of ES or iPS cells

Feeder-basedHuman• Cytokines,e.g.,FGF2• CryoStem™FreezingMedium• Smallmolecules

Mouse• Cytokines,e.g.,LIF• Smallmolecules

Feeder-freeHuman• NutriStem™Medium• Cytokines,e.g.,FGF2• Smallmolecules• CryoStemFreezingMedium

Samplepreparation Cellseparation Cellanalysis Cellculture Molecularapplications Clinicalapplications Samplepreparation Cellseparation Cellanalysis Cellculture Molecularapplications Reprogramming

Embryonic and induced pluripotent stem cellsPioneering solutions for ES and iPS cells research

Hematopoietic stem cellsMaking headway in your HSC research

iPS cells

Do you differentiate your ES or iPS cells?

Characterization of reprogrammed cells

Human• ExtracellularPSCstaining:TRA-1-60,TRA-1-81,SSEA-3,SSEA-4,CD326,CD324,etc.

• Extracellulardifferentiatedcellstaining:SSEA-1

Mouse• ExtracellularPSCstaining:SSEA-1• Extracellulardifferentiatedcellstaining:Anti-Feederantibodies

Isolation of reprogrammed cells

Magnetic isolationHuman• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads• Anti-SSEA-4MicroBeads• IsolationofTRA-1-81+cellsviaindirectmagneticlabeling

• hESCloningandRecoverySupplement

Flow sorting• Fluorochrome-conjugatedantibodies(markersabove)

Colony Picking• StainAlive™Antibodies

Generation of iPS cells

Human• mRNAReprogrammingReagents• Pluriton™ReprogrammingMedia• Smallmolecules

Mouse• ReprogrammingEcotropicRetrovirus• SmallMolecules

Differentiation of ES or iPS cells

Human or mouse• Cytokines• Smallmolecules

Purification of ES or iPS cells prior to differentiation

Human• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads

Mouse• PluripotentStemCellIsolationKit

Differentiation of ES or iPS cells via embryoid bodies

• EmbryoidBodyDissociationKit• gentleMACS™Dissociator

Monitoring differentiation by flow cytometry or immunohistochemistry

• Antibodies,forexample,useCD34antibodiestoanalyzecellsduringdifferentiationintohematopoieticorendotheliallineages.

Purification of cells during or after differentiation

MicroBeadreagents,forexample,cellseparationreagentsfordifferentiatedcells,e.g.,• NeuralCrestStemCellMicroBeads• CD34MicroBeads• CD144(VE-Cadherin)MicroBeads• CD309(Flk-1)MicroBeadKit• Anti-PSA-NCAMMicroBeadKit• Anti-A2B5MicroBeadKit

Sample type

PBMNCs or lysed whole blood• RedBloodCellLysisSolution

Isolation of HSCs

Magnetic isolation• LineageCellDepletionKit +CD117MicroBeads +Anti-Sca-1MicroBeadKit

(FITC)• CD105MultisortKit(PE)+Anti-Sca-1MicroBeadKit(FITC)

Flow sortingFluorochrome-conjugatedantibodiesagainst,e.g.,• Lineagemarkers• Anti-Sca-1• CD117(c-kit)• CD135(Flt3R)Isolation of HSCs

Magnetic isolation• CD34MicroBeadKit• CD133MicroBeadKit• LineageCellDepletionKit• CD117MicroBeadKit

Flow sortingFluorochrome-conjugatedantibodiesagainst,e.g.,• CD34• CD133• CD45• CD90• CD38

Expansion of HSCs

• Cytokines,e.g.,TPO,SCF,Flt3

Expansion of HSCs

• Cytokines,e.g.,SCF,Flt3

Phenotyping of HSCs

HumanFluorochrome-conjugatedantibodiesagainst,e.g.,• CD34• CD133• CD45• CD90• CD38• MACSQuant®FlowCytometers

MouseFluorochrome-conjugatedantibodiesagainst,e.g.,• Lineagemarkers• Sca-1• CD117(c-kit)• CD135(Flt3R)• MACSQuant FlowCytometers

Molecular analysis of HSCs

• mRNAIsolationKitandcDNASynthesisKit(forRT-PCR)

• ProteinIsolationKits(forChIPornormalIPs)

• Microarrayservices• SuperAmp™Technology• Bioinformatics• MultiMACS™M96Separator• Mitochondriaisolation

CFU assays for HSCs

Human• HSC-CFUMedia

Differentiation of HSCs

Human or mouse• Cytokines

Characterization of HSCs

ES cells

Transfection of ES or iPS cells

Human• Stemfect™hESCDNATransfectionKit• StemfectEnhancedhESCDNATransfectionKit• StemfectRNATransfectionKit• MACSelect™Products

Mouse• Stemfect2.0-mESCDNATransfectionReagent• MACSelect™Products

Single-cell passaging of ES or iPS cells

Human• hESCloningandRecoverySupplement• Anti-TRA-1-60MicroBeadKit• PluripotentStemCellMicroBeads

Monitoring of pluripotency by flow cytometry or immunohistochemistry

Human• ExtracellularPSCstaining:TRA-1-60,TRA-1-81,SSEA-3,SSEA-4,CD326,CD324,etc.• Extracellulardifferentiatedcellstaining:SSEA-1• Transcriptionfactorstaining:Oct4,Sox2,Nanog,etc.• MACSQuant®FlowCytometers

Mouse• ExtracellularPSCstaining:SSEA-1• Extracellulardifferentiatedcellstaining:Anti-Feederantibodies• Transcriptionfactorstaining:Oct4,Sox2,Nanog,etc.• MACSQuant®FlowCytometers

Translate research to clinical applications• GMPCytokines• CliniMACSSystem• CliniMACS®Reagents,e.g.,CD34,CD133

Removal of feeder cells

Depleting feeder cellsHuman• Anti-FibroblastMicroBeads

Mouse• FeederRemovalMicroBeads

Isolating pluripotent stem cellsHuman• Anti-TRA-1-60MicroBeadKit

• PluripotentStemCellMicroBeads

Culture of ES or iPS cells

Feeder-basedHuman• Cytokines,e.g.,FGF2• CryoStem™FreezingMedium• Smallmolecules

Feeder-freeHuman• NutriStem™Medium• Cytokines,e.g.,FGF2• Smallmolecules• CryoStemFreezingMedium

Sample type

• CD146• CD105• CD45

Isolation of MSCs

Expansion of MSCs

• CytoMix–MSC

Isolation of MSCs

Plastic adherence

Expansion of MSCs

inside!

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macs stem cell quick guide2013

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