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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) Cell separation with MACS® Technology MD0347.01 Miltenyi Biotec GmbH (headquarter) MACS® Magnetic Cell Sorting Magnetic labeling with MACS MicroBeads Elution of positively selected cells Flow through with unlabeled cells MD0651.01 MACS® Technology Equipment and reagents » MACS MicroBeads » MACS Columns » MACS Magnet MD0197.02 MiniMACS™ separator MD0343.02 Magnetic field generated in an MS Column MD0061.02 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 CD8+ T cells isolated by MACS® Technology MD0058.03 CD8+ T cells isolated by MACS® Technology MD0057.03 1/1 MD0xxx.01 MACS® isolated dendritic cell MD0159.02 Positive selection strategy Magnetic labeling with MACS® MicroBeads Elution of positively selected cells Flow through with unlabeled cells MD016102 Isolation of human NK cells from PBMC using CD56 MicroBeads CD56-FI TC 2.73% CD56-FI TC Positive fraction Relative cell number 17.86% Negative fraction Relative cell number Relative cell number Before separation 89.51% CD56-FI TC MD0158.02 Depletion strategy Magnetic labeling Isolation of untouched cells MD0160.02 Isolation of untouched B cells from PBMC using B Cell Isolation Kit Before separation Untouched B cells 95.86% CD19-PE CD19-PE 11.4% CD45-FITC CD45-FITC MD0147.02 Indirect MicroBeads Anti-Fluorochrome MicroBeads Anti-FITC, Anti-PE, Anti-APC Streptavidin MicroBeads Anti-Biotin MicroBeads Anti-Immunoglobulin MicroBeads MD0xxx.01 autoMACS™ Separator For automated cell separation including automated elution and system cleaning • Touch screen • Integrated computer • Uptake port • Elution ports • Solution bottles • Sample rack MD0423.02 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) 1/1 MD0xxx.01 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 MD0044.02 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 • µMACS mRNA Isolation Kits • µMACS One-step cDNA Kit Enrichment of transfected cells • MACSelect PIQOR™ gene expression profiling technology • Microarray & Bioinformatics Service cDNA Synthesis from small sample material 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 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.