Amber Dance is an award-winning freelance science writer based in Southern California. She is the ALS (Lou Gehrig’s disease) reporter for the Alzheimer Research Forum. She contributes to The Scientist and Nature journals, and has written about topics ranging from record-breaking rocks to bizarre new ant species.
Tissues are a complex mix of cell types, but it’s easy to pull out the ones you want to study. For some researchers, the key is fluorescence activated cell sorting (FACS), also called flow cytometry. But when quick, inexpensive cell purification is the goal, many turn instead to magnetic beads.
Magnetic beads can divide and capture a cell type of interest within a half-hour. Aided by antibodies to bind the desired cells within a mixed population, the beads stick to a magnet while all unbound cells remain free-floating.
The technique is popular for immunology and cancer studies, in which researchers often purify cells from blood or tissue samples of mice and humans. However, it can be used for just about any cell type, as long as you have the antibodies to match. For example, magnetic beads are used in food testing to capture potentially pathogenic bacteria. The technique also has clinical applications. Physicians at the University of Pennsylvania have used magnetic beads to isolate T cells from leukemia patients; they then modify these T cells to kill the cancer and return them to the patient.
The basic concept is “catch and release,” says Jeffrey Chalmers, a professor of chemical and biomolecular engineering at Ohio State University. First, you incubate the cells with antibodies to bind the surface of the cells. Then, you link the antibodies to the beads. This can be accomplished in several ways. The beads might be coated with secondary antibodies that grab onto the first set, for instance. Other kits use biotinylated primary antibodies and streptavidin, biotin’s favored binding partner, on the beads. In either case, the beads remain suspended in solution until placed in a magnetic field.
For many systems, the magnetic field is provided by a magnet in a test-tube holder. EasySep™ kits from STEMCELL Technologies and Dynabeads® from Life Technologies work in this way. The beads, and attached cells, stick to the tube’s sides, and you then pour or pipette out the rest. Another option, the MACS® from Miltenyi Biotec, runs the cells through a column instead of settling them in a test tube, but the principles are similar.
These systems support both manual and automated workflows.. STEMCELL’s RoboSep is an automated system, and Dynabeads work with several laboratory robots. MACS also is automatable, using, for instance, Miltenyi Biotec’s autoMACS® Pro Separator or MultiMACS™ Cell24 Separator.
Magnetic separation provides a simple alternative to FACS. The beads work hundreds or thousands of times faster, Chalmers notes, because they sort in bulk instead of considering each individual cell. And the equipment is relatively cheap compared to a cytometer.
However, the typical magnetic-sorting protocol cannot match FACS’ purity. Also, magnetic systems usually only separate cells based on a yes or no question—the antibody binds, or it doesn’t. FACS can complete more complex sorts, taking into account multiple markers and varying expression levels.
Depending on your needs, there are different approaches to sort the cells you’re after.
Positive selection
This is the simplest approach: You use one antibody for the cell type you want to keep. For example, HIV researchers might want to isolate CD4+ T cells with a CD4 antibody. With positive selection, one can achieve 98% or higher purity, says Vicki Stronge, senior product manager for immunology at STEMCELL Technologies. It’s particularly well suited for rare cell types, she adds.
The downside of positive selection is that the cells you wanted are now stuck to antibodies, which are stuck to magnetic beads. Some researchers worry that the cells will internalize the smaller, nanoscale beads and suffer ill effects. The particles may persist inside cells for days, says Axl Neurauter, a research manager at Life Technologies’ Oslo, Norway, office, who works on Dynabeads. However, there is little research to show that the ingested particles are truly toxic. Using larger, micron-sized beads alleviates this concern. Dynabeads also are coated with a polymer shell to isolate the metal of the beads from the cells.
Another concern is that bead-cell attachment could interfere with downstream experiments. For example, if you want to examine gene expression, the binding of a cell-surface receptor to an antibody might alter the transcriptome, Neurauter says.
However, the beads don’t interfere with many assays, such as gel electrophoresis. There also are methods to uncouple the cells from the beads. For example, with Dynabeads linked to cells by biotin-streptavidin binding, you can add excess biotin to outcompete the biotin on the cells, releasing them from the streptavidin on the beads.
Negative selection
To keep the desired cell type pristine, you can perform negative selection, which for many scientists has become the preferred approach, Stronge says. In the CD4-cell example, this would mean incubating the cell mix with antibodies for every cell type except CD4 cells. The unwanted cells stick to the tube’s walls, and you can remove the purified population. Negative selection is useful when the cell of interest has no unique markers, Neurauter says. It yields about 95% purity, according to Stronge.
Companies that make magnetic-separation kits provide pre-mixed antibody cocktails to perform negative selections for standard cell types. For example, you can get EasySep kits for more than three-dozen negative selections, such as human basophils or mouse-mammary stem cells. You also can create your own antibody mix or work with the manufacturer on a custom cocktail.
Depletion
Depletion is a special case of negative selection in which you wish to get rid of one cell type and keep everything else. For example, Stronge says, vaccine researchers often want to focus on how CD4+ cells interact with other types but eliminate the CD8+ cells, because their functions overlap with those of CD4+ cells. Depletion works like negative selection, but with a single antibody.
Magnetic separation is fairly straightforward, but there are things you can do to optimize your results. For the average user, the quality of the antibody is the top concern, Chalmers says. It’s also important to prepare samples without any cell clumps and with the right concentration of cells for the apparatus, recommends Stronge. “The better you can do the sample preparation, the better the result is going to be at the end,” she says.
The image at the top of the page is from Life Technologies.