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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)
MD0347.01
Cell separation with MACS® Technology
Miltenyi Biotec GmbH (headquarter)
MD0651.01
MACS® Magnetic Cell Sorting
Magnetic labeling with
MACS MicroBeads
Flow through with unlabeled cells
Elution of positively selected
cells
MD0197.02
MACS® Technology
Equipment and reagents
» MACS MicroBeads
» MACS Columns
» MACS Magnet
MD0343.02
MiniMACS™ separator
MD0061.02
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
MD0058.03
CD8+ T cells isolated by MACS® Technology
MD0057.03
CD8+ T cells isolated by MACS® Technology
1/1 MD0xxx.01
MD0159.02
MACS® isolated dendritic cell
MD016102
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
MD0158.02
17.86% 2.73% 89.51%
MD0160.02
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
MD0147.02
11.4% 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
MD0423.02
1/1 MD0xxx.01
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
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
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