1. 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

2. 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

3. 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 !

4. 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

5. 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

6. 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

7. 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

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

9. 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

10. 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^ 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

11. 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

12. 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