Magnetic isolation of cells molecules with MACS

Magnetic isolation of cells & molecules with MACS® Technology

Thuesday: MACS® Technology for magnetic isolation of cells and molecules • Introduction • Features of paramagnetic Micro. Beads • 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 MD 0347. 01

Miltenyi Biotec Gmb. H (headquarter)

MACS® Magnetic Cell Sorting Magnetic labeling with MACS Micro. Beads Elution of positively selected cells Flow through with unlabeled cells MD 0651. 01

MACS® Technology Equipment and reagents » MACS Micro. Beads » MACS » Columns MACS Magnet MD 0197. 02

Mini. MACS™ separator MD 0343. 02

Magnetic field generated in an MS Column MD 0061. 02

Major advantages of MACS® Micro. Beads • 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

CD 8+ T cells isolated by MACS® Technology MD 0058. 03

CD 8+ T cells isolated by MACS® Technology MD 0057. 03

1/1 MD 0 xxx. 01

MACS® isolated dendritic cell MD 0159. 02

Positive selection strategy Magnetic labeling with MACS® Micro. Beads Elution of positively selected cells Flow through with unlabeled cells MD 016102

Isolation of human NK cells from PBMC using CD 56 Micro. Beads CD 56 -FI TC 2. 73% CD 56 -FI TC Positive fraction Relative cell number 17. 86% Negative fraction Relative cell number Before separation 89. 51% CD 56 -FI TC MD 0158. 02

Depletion strategy Magnetic labeling Isolation of untouched cells MD 0160. 02

Isolation of untouched B cells from PBMC using B Cell Isolation Kit Before separation Untouched B cells 95. 86% CD 19 -PE 11. 4% CD 45 -FITC MD 0147. 02

Indirect Micro. Beads Anti-Fluorochrome Micro. Beads Anti-FITC, Anti-PE, Anti-APC Streptavidin Micro. Beads Anti-Biotin Micro. Beads Anti-Immunoglobulin Micro. Beads MD 0 xxx. 01

auto. MACS™ Separator For automated cell separation including automated elution and system cleaning • Touch screen • Integrated computer • Uptake port • Elution ports • Solution bottles • Sample rack MD 0423. 02

The Clini. MACSTM System • CD 34+ cell selection: hematopoetic precursor cell • CD 133+ cell selection: (hematopoetic) stem cell Depletion of T cells (allogenic, Gv. HD) or tumor cells (autologous) 1/1 MD 0 xxx. 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 MD 0044. 02

MACSmolecular Specific protein isolation • µMACS Protein A and Protein G Micro. Beads for immunoprecipitation • µMACS Epitope Tag Protein Isolation Kits • µMACS Streptavidin Kit m. RNA isolation & c. DNA synthesis • µMACS m. RNA Isolation Kits • µMACS One-step c. DNA Kit Enrichment of transfected cells • MACSelect PIQOR™ gene expression profiling technology • Microarray & Bioinformatics Service

c. DNA Synthesis from small sample material 500 cells, µMACS One-step c. DNA 500 cells m. RNA + RT in tube* 5 cells, µMACS One-step c. DNA 5 cells, m. RNA + RT in tube* Negative control Jurkat cells *m. RNA + RT in tube: competitor (magnetic) m. RNA isolation kit and conventional in -tube synthesis Light. Cycler quantitative PCR with intron-spanning primers for 87 bp GAPDH fragment, 10 and 20 % of each c. DNA was used.