Description
Background information
The MACSelect LNGFR - Transfected Cell Selection Kit contains the vectors pMACS LNGFR-IRES and pMACS LNGFR, a control vector, MACSelect LNGFR MicroBeads, Anti-LNGFR-FITC antibody for detection of cells before enrichment, MACSelect Control FITC antibody for detection of cells after enrichment, and a CD14-FITC antibody for analysis of the control experiment when doing a co-transfection. All components are also available separately. The pMACS LNGFR-IRES vector enables the bicistronic expression of the gene of interest from a CMV promotor and the truncated LNGFR molecule via an internal ribosome entry side (IRES). The pMACS LNGFR vector encodes the truncated LNGFR marker and is recommended for co-transfection in combination with an expression vector encoding the gene of interest. Transfected cells are magnetically labeled with MACSelect LNGFR MicroBeads and subsequently separated from non-transfected cells.
Note: The LNGFR molecule is expressed on cells of the central and peripheral nervous system, on bone marrow fibroblasts, follicular dendritic cells, and some mesenchymal cells. Therefore, the alternative MACSelect 4 or MACSelect Kk Systems should be used instead of the MACSelect LNGFR System when working with these cells.
Detailed procedure
Cells are transfected with the transfection method of choice using either the pMACS LNGFR.IRES vector harboring the gene of interest for single vector transfection or with the pMACS LNGFR vector and an expression vector encoding the gene of interest. The time of optimal marker expression, which determines the incubation time post transfection, has to be taken into consideration for each individual experiment. The level of marker expression can be determined by staining an aliquot of transfected cells with the Anti-LNGFR-FITC antibody followed by fluorescence microscopy or flow cytometric analysis. Depending on the cell type, transfection method, and culture conditions cells generally need to be cultured between 6 and 84 hours. After incubation cells are harvested and magnetically labeled using MACSelect LNGFR MicroBeads. Cells are loaded onto an MS or LS Column placed in a MiniMACS or MidiMACS Separator. After washing, only the magnetically labeled transfected cells are retained on the column. Magnetic separation can also be carried out on the autoMACS Pro Separator. After enrichment, cells can be analyzed by fluorescence microscopy or flow cytometry using the supplied MACSelect Control FITC antibody.
Applications
The MACSelect LNGFR System facilitates the enrichment of a homogeneous cell population of transiently transfected cells eliminating the need for setting up stable cell lines. As the MACSelect LNGFR MicroBeads are non-toxic, the isolated cells can be directly cultured. Stable cell lines can also be rapidly and efficiently established by repeated magnetic enrichment without the need for toxic antibiotic treatment. The system can be used for the enrichment of transiently transfected or transduced primary cells to a purity that enables sensitive downstream analysis. It is suitable for transfected cell selection when a rapid, non-toxic and non-immunogenic method is required. Additionally, the system can be applied for selection of transduced T cells, other hematopoietic cells, or stem cells for research on allogeneic hematopoietic stem cell transplantation.
Downstream applications
After enrichment the transfected cells can be subjected to flow cytometry, fluorescence microscopy, (co-)immunoprecipitation experiments, Western blot analysis, or reporter gene assays in functional gene and drug screening, signal transduction, protein interaction, and RNAi knockdown studies.
Overview
The MACSelect LNGFR System uses the transiently expressed truncated human low-affinity nerve growth factor receptor (LNGFR) molecule as a surface marker to select transfected cells. It can be used in virtually all LNGFR-negative adherent or suspension cell lines and primary cells in combination with common transfection methods.
