BioLegend, Inc
Senior Product Manager
The separation of specific cells within a complex cell mixture is a challenge that researchers face on a daily basis. Three major techniques have emerged as most widely used: centrifugation-based, fluorescence-activated cell sorting (FACS), and magnetic cell separation. Centrifugation is the least expensive, but is also the least specific, with separation based largely on the cells’ physical properties (density) and limited ability to separate cells based on their phenotype.
FACS and magnetic cell separation are far more specific than centrifugation, as the methodologies utilize conjugated antibodies that specifically recognize the target cells. Using FACS, cells are separated based on the expression of several antigens simultaneously. Magnetic separation is limited to one or two antigens, but has multiple advantages over FACS, including a much shorter processing time, a simpler platform, and significantly lower cost. As magnetic separation is becoming a more widely adopted technique, here are four important points to consider when performing this method.
Sample composition
If you know the components of the sample mixture you are working with, this translates into a better planned and well-executed experiment. This may be especially important in magnetic cell separation, where familiarity with the different cell populations in your mixture can help you maximize the purity and yield of your isolation. It is not uncommon to compare two or more groups; for example, one control and one treated. The treated group can end up having a different percentage of target cells. Knowing this in advance allows you to adjust the reagents to achieve similar purity in the two groups. The same applies in the case of up or down-regulation of target markers. If you are using a marker for positive selection and its expression changes after treatment, it may be best to choose an alternative marker, if available. Or you can choose a kit that utilizes a negative selection procedure.
Positive or negative selection, which way to go?
In general, there are two strategies for magnetic cell separation: positive and negative selection. For positive selection, it is most common to use magnetic particles conjugated to antibodies that directly bind the target cell. For negative selection, usually an antibody cocktail binds the majority of the cells that are not the target, followed by magnetic particles that capture the antibodies. The cells of interest are collected in a new, clean tube, untouched by any reagent. The choice between positive or negative selection depends on your specific needs, but some factors to take into account are:
- Positive selection is generally faster. The use of antibody-conjugated beads allows for a single incubation step and less washing. However, more efficient, no-wash protocols for negative selection are becoming standard.
- Enrichment step prior to FACS. It is not unusual to combine more than one technique to purify complex populations or more than one cell type at the same time. An efficient way to achieve this is to use positive selection followed by FACS. For example, for low frequency targets such as mouse dendritic cells (DCs), a good approach to purify more than one subset is to enrich the total DC fraction using CD11c-conjugated beads and further sort by FACS the CD8+ and CD11b+ subsets, or other relevant subsets present in your sample.
- Functional studies. When doing positive selection, the isolated cells will carry the conjugated beads, unless they can be removed using a secondary reagent or treatment. In most cases, this is not an issue. However, if the clone used to select the cells has blocking or agonistic properties, or you have determined that positively selecting your cells may have an impact in your experimental readout, negative selection may be the best way to go.
Calculate purity and yield
After you have isolated your cells, you may need to verify the purity of the resulting cell preparation. Purity is defined as the percentage of target cells collected, which can be done by flow cytometry. Use a viability dye to exclude dead cells from the analysis. For positive selection, it may be advisable to use a compatible clone with your antibody-conjugated beads. Suppliers should advise you on a safe combination to use, so check with the manufacturer if needed. For negative selection, there is no restriction, as the isolated cells have no reagents bound on the surface.
The yield is defined as the percentage of recovered target cells relative to the number of those cells present in your sample in the original cell suspension. Manufacturers and researchers may define this parameter slightly differently, but there are ways to help equalize this across multiple users. One would be to determine the number of target cells right before applying the sample to the magnetic field, and right after final collection from the magnet. This reports the yield of the magnetic separation only. The other way is to calculate yield for the entire process. For this, determine the number of cells in the initial cell suspension and after collecting the target population as ready to use. The number of cells can be determined by flow cytometry. Independently of which way you decide to calculate the yield, it will always be reported as number of sorted target cells/number of target cells before sorting. Do not use number of total cells in the denominator, as that wouldn’t be the accurate yield.
The sweet spot, balancing between purity and yield
An inherent property of magnetic isolation is maintaining the relationship between purity and yield. As you work to increase the purity, the yield decreases, and vice versa. So the art lies in balancing the factors to obtain the optimal purity and yield. With BioLegend’s magnetic separation reagents, to maximize the purity for positive selection, there are two options to try: titrate down the volume of nanobeads and/or increase the number of separations with the magnet. For negative selection, you can increase the volume of streptavidin nanobeads and perform a single separation. To maximize yield (e.g., if you are sorting by flow afterwards), just do the opposite. For example, for positive selection, increase the volume of beads used and/or decrease the number of separations.
Optimizing a cell separation experiment is challenging, especially when using custom reagents. However, applying the tips discussed here may help you obtain the best results possible. From sample preparation to downstream applications, the keys to success are sample characterization, strategy, reagent selection, and protocol optimization.
Image: Courtesy of BioLegend.
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