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)

MD Cell separation with MACS ® Technology

Miltenyi Biotec GmbH (headquarter)

MD MACS ® Magnetic Cell Sorting Magnetic labeling with MACS MicroBeads Flow through with unlabeled cells Elution of positively selected cells

MD MACS ® Technology Equipment and reagents » MACS MicroBeads » MACS Columns » MACS Magnet

MD MiniMACS ™ separator

MD 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

MD CD8 + T cells isolated by MACS ® Technology

MD CD8 + T cells isolated by MACS ® Technology

1/1 MD0xxx.01

MD MACS ® isolated dendritic cell

MD Positive selection strategy Magnetic labeling with MACS ® MicroBeads Flow through with unlabeled cells Elution of positively selected cells

Isolation of human NK cells from PBMC using CD56 MicroBeads Before separation CD56-FITC Relative cell number Negative fraction CD56-FITC Relative cell number Positive fraction CD56-FITC Relative cell number MD %2.73%89.51%

MD Depletion strategy Magnetic labeling Isolation of untouched cells

Isolation of untouched B cells from PBMC using B Cell Isolation Kit Before separation CD45-FITC CD19-PE Untouched B cells CD45-FITC CD19-PE MD %95.86%

MD0xxx.01 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 MD

1/1 MD0xxx.01 The CliniMACS TM 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 MD

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