James Netterwald, PhD, is a freelance science and medical writer based in New Jersey. His writing serves every life science industry.
Isolation of a genetically homogeneous population of cells is a key step in many cell-based, downstream applications, including gene characterization studies, host-pathogen interaction studies, cell counting, biochemical analysis, protein isolation, cell-cell interactions and more.
These applications often begin with a cell-isolation kit. The critical reason to use a cell-isolation kit is to ensure that the results of such applications accurately depict the biology of the targeted cell type, not a population of cell types. There are several cell-isolation kits available for purchase. The various kits differ by isolation method and selection marker; the following are key points to consider.
Positive vs. negative selection
Cell-isolation kits are based on one of two principles: positive selection or negative selection. Kits that work by positive selection capture the target cells directly via the binding of the cells of interest to an antibody. This antibody is most often conjugated to a magnetic bead. The antibody-bead-cell complexes are then removed from suspension by a magnet, and the beads are removed from the cells of interest with a secondary antibody. In contrast, kits that work by negative selection capture target cells indirectly by first depleting the sample of all other unwanted cells—using the same antibody-coated bead method—thus leaving behind only the target cells.
The choice to use a positive or negative selection method depends on whether or not there is a robust selection marker present on the surface of the target cell. A robust selection marker can enable the specific capture of a target cell without contamination by non-target cells. For target cells that do have a robust selection marker, it is best to choose a kit that works by positive selection. In contrast, for target cells that do not have a robust selection marker, you’re better off with a kit that works by negative selection.
Choosing your selection marker
Literature research, using databases such as PubMed, is the first step in determining which selectable markers are expressed on your cells of interest. If the literature search does not identify suitable surface markers, conduct a BLAST search of your target cell’s genetic code (assuming you know the DNA sequence) to identify the cell-surface proteins that might be used to capture your cell population. Your BLAST search should reveal which surface markers are potentially expressed on your cells of interest. A BLAST search on a T cell sequence would reveal whether a given cell of interest expresses CD4 or CD8, for example. If you find that your cell of interest expresses CD4, perform an immunoassay (for example, ELISA or immunoagglutination assay) on a heterogeneous sample of your cell source (for example, spleen-tissue lysate) to confirm the BLAST-search result. After the data have been confirmed, you can choose a cell-isolation kit that selects T cells that express the CD4 marker.
A typical workflow
In a typical kit designed to isolate total CD4-positive T cells, for example, the cell source is first incubated with superparamagnetic, anti-CD4 antibody-coated beads. The antibody binds to the CD4+ T cells, and the beads are then washed to remove unwanted (i.e., non-CD4 expressing) cells. This step is followed by incubation with a secondary antibody directed against the anti-CD4 antibody on the beads. This binding displaces the CD4+ cells, and the bead-antibody-antibody complexes are then removed by a magnet, leaving the supernatant, which contains antibody-free, bead-free, total CD4+ T cells, which can be poured off into a new tube and used in the application of your choosing.
Whatever the downstream application, your results can only be as good as your starting material. By carefully choosing your selection method and the marker you will select with, you can wind up with a pure population of cells and be well on your way to some beautiful results.
The image at the top of the page is from StemCell Technologies.