Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
If you are interested in cell sorting, you may be seeking some clarification regarding the different types of sorting. There are two main types of cell sorting used to separate cells into different populations—flow cytometry and magnetic bead separation. Newer sorting techniques are in development and will soon add variety to these old standards. A major difference between these methods is that flow cytometry sorts cells one by one, while magnetic bead separation works on all cells at once. Although both methods are efficient, knowing their relative strengths and weaknesses can help you make an informed choice.
Sorting by flow cytometry
How it works. The commonly-used sorting powerhouse of flow cytometry is known as fluorescence activated cell sorting (FACS). In FACS, cells are funneled single-file through a narrow opening that ends in a nozzle, such that droplets of fluid emerge one at a time. Each droplet may, or may not, contain one cell. As the droplet falls, it passes through a laser (or several lasers). If the cell is labeled with a fluorescent tag that is excited by the laser light, the fluorescent signal it subsequently emits will be noted by detectors. The scatter of the laser light and the fluorescent signal tell a computer which (pre-specified) population each cell belongs to. The droplet is collected, and the computer directs the FACS instrument to send the droplet to the appropriate location. For example, the computer may sort droplets into categories of no cell, cell with no fluorescent signal, cell with a green fluorescent signal, cell with a red fluorescent signal and cell with both green and red fluorescent signals.
What it’s best for. FACS is best when you want your sorted cell population to have a higher purity and recovery (other methods give lower recovery, possibly because of cell mortality or cell loss during washing steps). It is also best when you are sorting based on an intracellular characteristic (to which magnetic beads would not have access); FACS can sort cells labeled with fluorescent probes for nuclear or other intracellular targets.
FACS is also best if you need information about cell surface molecules, such as membrane protein receptors. When labeled with specific fluorescent markers, FACS can sort such receptors even if they are especially low in density. And it can sort cells according to presence, absence and density of the receptors.
Sorting with magnetic beads
How it works. Magnetic bead separation is a bulk separation method that's also known as immunomagnetic cell sorting, or MACS. Your starting sample of cells is incubated with tiny magnetic beads that have been attached to specific antibodies. Magnetic force is used to collect the beads and anything bound to them, and the free material is washed away. This method can be used either to retain your cells of interest or to pull out unwanted cells, leaving behind a purer sample of your cells of interest.
What it’s best for. Sorting cells using magnetic beads is appropriate when you are separating according to only one separation criterion or characteristic, rather than several. It is also best as a bulk method that classifies and sorts simultaneously (in contrast to FACS, which classifies and sorts sequentially). Magnetic beads separation is a particularly good, and often-used, preparatory step prior to FACS.
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Some cell types exhibit intrinsic cell fluorescence, which would disrupt the ability of a FACS instrument to detect signals. In this situation, sorting with magnetic beads would be the best option.
Sorting on microchips
How it works. Using microchips is a newer version of cell sorting pioneered by microfluidics researchers. A tiny sample is injected into a microfabricated chip containing microfluidic channels. The sample fluid moves through the tiny channels, pneumatically controlled by pumps and valves. Cells pass single-file through a laser in a sorting area, and the scattered and fluorescent light signals are collected. Pumps can re-route the direction of travel for individual cells, for example to send them to a “waste” channel or a “keep” channel. Or it can send them back into the sorting line to be analyzed again for verification.
What it’s best for. Sorting this way is best when you want to use extremely small sample volumes, perhaps because the sample material is scarce, precious or extremely expensive. Microfluidic-based cell sorters are like tiny, simplified FACS instruments in that they sort cells singly. Compared with FACS, microfluidic chips may experience lower background fluorescence, although they also have a lower recovery rate (but keep in mind, the starting sample volume is much smaller).
Keep your eyes open for further innovations in cell sorting techniques—but in the meantime, enjoy the reliability of the old standbys.
The image at the top of the page is from the BD Accuri C6 flow cytometer.