Magnetic isolation

of cells & molecules

with MACS® Technology

Thuesday:MACS® Technology for magnetic isolation of cells and molecules

• Introduction • Features of paramagnetic MicroBeads • General procedure of magnetic cell isolation • Overview of applications in molecular biology

µMACS epitope tagged protein isolation • Expression of tagged/fusion proteins, e.g. GFP fusion proteins • Magnetic protein isolation

Wednesday: • Detection of proteins, results and trouble-shooting • Optimizing protein expression analysis by transfected cell selection (MACSelect)

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Cell separation with MACS® Technology

Miltenyi Biotec GmbH (headquarter)

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MACS® Magnetic Cell Sorting

Magnetic labeling with

MACS MicroBeads

Flow through with unlabeled cells

Elution of positively selected

cells

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MACS® Technology

Equipment and reagents

» MACS MicroBeads

» MACS Columns

» MACS Magnet

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MiniMACS™ separator

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Magnetic field generated in an MS Column

Major advantages of MACS® MicroBeads

• Extremely small beads (50 nm Ø) => Colloidal suspension, non-sedimenting

=> fast reaction kinetics => short incubation times • Super-paramagnetic • Biodegradable & non - toxic => Straight to experiment or cell culture

=> Cell function preserved

• Flow cytometry compatible=> No bead detachment required

=> Only 20-30% of binding sites occupied

=> Decreased flow sorting time +MACS® separation

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CD8+ T cells isolated by MACS® Technology

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CD8+ T cells isolated by MACS® Technology

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MACS® isolated dendritic cell

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Positive selection strategy

Magnetic labeling with

MACS® MicroBeads

Flow through with unlabeled cells

Elution of positively selected

cells

Isolation of human NK cellsfrom PBMC using CD56 MicroBeads

Before separation

CD56-FITC

Rel

ativ

e ce

ll n

um

ber

Negative fraction

CD56-FITC

Rel

ativ

e ce

ll n

um

ber

Positive fraction

CD56-FITC

Rel

ativ

e ce

ll n

um

ber

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17.86% 2.73% 89.51%

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Depletion strategy

Magnetic labeling Isolation of untouched cells

Isolation of untouched B cellsfrom PBMC using B Cell Isolation Kit

Before separation

CD45-FITC

CD

19-P

E

Untouched B cells

CD45-FITC

CD

19-P

E

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11.4% 95.86%

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Indirect MicroBeads

Anti-Fluorochrome MicroBeads Anti-FITC, Anti-PE, Anti-APC

Streptavidin MicroBeads Anti-Biotin MicroBeads

Anti-Immunoglobulin MicroBeads

For automated cell separation including automated elution and system cleaning

autoMACS™ Separator

• Touch screen

• Integrated computer

• Uptake port

• Elution ports

• Solution bottles

• Sample rack

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The CliniMACSTM System

• CD34+ cell selection:hematopoetic precursor cell

• CD133+ cell selection:(hematopoetic) stem cell

Depletion of T cells(allogenic, GvHD) or tumor cells(autologous)

MACS® separation strategies: NO LIMITS!

• Positive selection and depletion

• Isolation of cell subsets

• Isolation using any antibody

• Isolation and detection of secreting cells

• Isolation of molecules

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MACSmolecular

Specific protein isolation• µMACS Protein A and Protein G MicroBeads for

immunoprecipitation• µMACS Epitope Tag Protein Isolation Kits• µMACS Streptavidin Kit

mRNA isolation & cDNA synthesis

Enrichment of transfected cells

• µMACS mRNA Isolation Kits • µMACS One-step cDNA Kit

• MACSelect

PIQOR™ gene expression profiling technology• Microarray & Bioinformatics Service

cDNA Synthesis from small sample material

Jurkat cells

*mRNA + RT in tube: competitor (magnetic) mRNA isolation kit and conventional in-tube synthesis

LightCycler quantitative PCR with intron-spanning primers for 87 bp GAPDH fragment, 10 and 20 % of each cDNA was used.

500 cells, µMACS One-step cDNA500 cells mRNA + RT in tube*5 cells, µMACS One-step cDNA5 cells, mRNA + RT in tube*Negative control