In several of our blogs, we have discussed the power of flow cytometry to identify unique cell populations, both rare and abundant. Flow cytometry also offers the opportunity to actually sort out the cells of interest for a variety of downstream applications. Fluorescence Activated Cell Sorting (FACS) and immunomagnetic cell sorting (MACS) are two of the most widely used methods for the isolation of phenotypically identified cells. Read more to make sense of FACS, MACS, and finding the best separation strategy for your needs.
The FACS approach starts with individual fluorescently labeled cells being streamed passed a laser in a fluid droplet. If the fluorescent label on the cell is excited by the laser, a computer will collect this data and specific types of cells will be deflected into a collection tube based on predetermined criteria. These populations can reach a purity of 95% or greater and are considered a “pure” cell population of a defined phenotype for downstream studies such as genomic profiling or functional analysis. Cell sorters can now separate multiple different cell populations using different lasers and a variety of fluorescent labels and are optimal for applications needing high levels cell population purity. FACS protocols are flexible and can be fine-tuned to collect your cell population based on different parameters for purity or speed of collection. Rare cell type isolation offers challenges for both purity and recovery so the best path is to work with very experienced professionals. Cells going through cell sorters also undergo stress traveling through the high pressure fluidics system of the instrument, so care must be taken to preserve viability and functionality.
For MACS, cells are incubated with immunomagnetic beads coated with specific antibodies, and a magnet is applied to the sample to mechanically separate cells that are bound to the beads from unbound cells. MACS can be used in two different ways to separate cells: positive selection by which your cells of interest are bound to the bead and then subsequently washed off to recover your pure population, or negative selection by which cells you are excluding are bound to beads and your cells of interest are unbound and can be recovered. MACS is an effective technique for large-volume bulk cell separations and can be used with different immunomagnetic beads in series to optimize selection. However, the resulting sorted samples obtained by MACS is often not as high in purity as with FACS, and MACS technology is limited if the phenotype depends on multiple antibodies.
MACS separation prior to FACS is a widely used approach to enrich populations that can be further separated by FACS to optimize the yield of rare cell populations. This combination of approaches often improves both yield and speed of sorting.
Making a Decision
The best sorting strategy for you comes down to your needs. Consider the downstream analysis you will be doing, the level of purity you need, and the sorting equipment you have access to as you determine the technique that will work best for you.