MACS® Technology for magnetic isolation of cells and molecules 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) 1

Protein expression and isolation in eukaryotic cells - critical points transfection of eukaryotic cells & percentage transfected versus untransfected cells protein expression level lysis of cells protein localisation e.g. membrane protein, nuclear protein sensitivity of detection specificity of detection

Eukaryotic cell transfection Challenges • difficult to transfect cells like primary cells, suspension cells • background of untransfected cells (reduced sensitivity of analysis) => Enrichment of transfected cells Antibiotic selection • possible effects of antibiotica on cell viability and function • needs time (several days to weeks) or... MACSelect !

Principle of MACSelect transfected cell selection: co-expression of surface marker Plasmid with DNA of interest pMACS Co-transfection or single vector transfection Incubation Magnetic labeling Magnetic separation Enriched transfected cells

MACSelect surface markers NEW H-2Kk CD4 LNGFR • trypsin sensitive • requires co-expression of beta-2-microglobulin • CD4 is naturally expressed on T helper cells, monocytes and dendritic cells • H-2Kk expression is restricted to some rarely used mouse strains like AKR/J and CBA/Ca. • LNGFR is expressed in the CNS and PNS on autonomic and sensory neurons and glial cells, on bone marrow fibroblasts, follicular dendritic cells, and some mesenchymal cells

Example: 1881 cell enrichment with pMACS LNGFR-IRES-CD14 and MACSelect LNGFR Transfection (ori) After enrichment (pos) 5.2 % 80.7 % MACSelect Control 79.7 % 5.2 % 1881 cells were transfected with pMACS LNGFR-IRES-CD14 and separated 18 hours later. Labeling with MACSelect LNGFR MicroBeads (before enrichment: ori, after enrichment: pos), enrichment with MS Column. Staining with MACSelect Control FITC or CD14.PE. Transfected cells were enriched from 5% to approx. 80% after one round ofselection. With a second column, enrichment was improved to >95% (data not shown). CD14 (gene-of-interest) forward scatter forward scatter

Enrichments of transfected CHO cells with MACSelect 4 This is an example of a MACSelect enrichment after a very low transfection efficiency of less than 4% transfected CHO cells. After enrichment with MACSelect CD4 MicroBeads more than 90% of transfected cells were obtained. Cells are stained with MACSelect control/CD4 FITC antibody and a nuclear counterstain to show the enrichment. Transfection of CHO cells with pMACS 4-IRES.II, magnetic labeling with MACSelect 4 MicroBeads and separation, staining with MACSelect Control FITC Antibody/CD4-FITC Antibody, nuclear counterstain with Toto3 <4% transfected cells >90% transfected cells after enrichment

MACSelect Transfected Cell Selection Kits • Transfect difficult cells (<5% pos. cells) • Gentle cell enrichment - no antibiotics • Fast - 3-4 hours • No change of transfection method • For any cell type

Co-expression of epitope-tagged BDCA-2 and surface selection marker Co-transfection + BDCA-2.PE del-H-2Kk HA-BDCA-2 Two constructs were used: a c-myc-tagged BDCA-2 expressed with the new MACSelect LNGFR marker in a bicistronic sequence directly coupling BDCA-2 expression to LNGFR expression, and a hemagglutinin-tagged BDCA-2, coexpressed with the MACSelect H2-Kk marker. All surface markers used have truncated cytoplasmatic domains. A double labeling with LNGFR- or H2KK-FITC and BDCA-2-PE shows a high number of untransfected or low expressing cells and a low fraction of cells strongly expressing BDCA-2. H-2Kk.FITC © Miltenyi Biotec

Enrichment of BDCA-2+/H-2Kk+ cells from 108 total cells using MACSelect Kk MicroBeads Before enrichment SSC Here you can see the result of the MACSelect enrichment starting from a total number of 10^8 1881 cells with different amounts of transfected cells. The small fraction of BDCA-2 positive cells is enriched after MACSelect; even with as low as 0,1% BDCA-2 transfected cells after you can isolate a cell fraction with a high number of BDCA-2 positive cells*. * 40%, 47%, 55% purity, depending on gate set even higher After enrichment anti-BDCA-2.PE © Miltenyi Biotec

HA tagged control protein Isolation of HA-BDCA-2 using anti-HA MicroBeads _ _ Cell isolation: MACSelect µMACS Anti-tag µMACS Anti-tag _ Protein isolation: HA tagged control protein 0.1% 1% 10% 0.1% 1% 10% 0.1% 1% 10% % BDCA-2 pos. cells: HA-BDCA-2 This is the result of the BDCA-2 expression: The picture on the left side shows a western blot with an anti-HA antibody (+ anti-mouse-HRP) and peroxidase catalysed staining. Without transfected cell and protein isolation hardly any BDCA-2 can be detected on the western blot. With µMACS HA-Tag protein isolation a strong protein band is visible. The extended band is due to glycosylation of BDCA-2 resulting in different sizes. Additional bands show a dimer, signals below belong to BDCA-2 degradation products. The combination of MACSelect and µMACS protein isolation gives even stronger bands. Now you don‘t have to scale up transfection and immunoprcipitation in case of low transfection efficiencies and low protein expression. (10^7 cells each, no isolation: lysis in 50µl, 1/5 on gel; Tag-isolation: 1/5 eluate; MACSelect+Tag isolation: 10^8 cells transfected, approx. 50% => Tag isolation from 5x10^7 cells (HA-tag control protein: 8.5ng, 17.5ng, 51ng; ECL develop ~1min; no antibody light chain visible- only with very long exposure) The x-ray picture on the right hand side shows the specific isolation of c-myc-BDCA-2, that is not detectable with HA-Tag isolation. © Miltenyi Biotec

Protein expression and isolation in eukaryotic cells - critical points transfection of eukaryotic cells & percentage transfected versus untransfected cells protein expression level lysis of cells protein localisation e.g. membrane protein, nuclear protein sensitivity of detection specificity of detection