Secondary Antibodies for Flow Cytometry

Flow cytometry is a technique for identifying specific cell types within a heterogeneous population. It involves incubating a single-cell suspension with fluorophore-labeled antibodies for cellular markers before introducing the sample into the flow cytometer. Here, a stream of fluid directs the cells in single file past an interrogation point where one or more lasers are focused. As each cell crosses the interrogation point, the lasers excite the bound fluorophores, causing them to emit fluorescence. The resultant signals are subsequently deconvoluted and used for sample characterization. A flow cytometry experiment can employ either direct or indirect detection depending on the number of markers being investigated. When a large number of markers is required for cellular identification, direct detection with fluorophore-labeled primary antibodies is more common. This is because direct detection simplifies panel design by allowing for multiple antibodies from the same host species to be combined in a single experiment. A further advantage of direct detection is that it shortens the experimental workflow by eliminating the need for a secondary antibody incubation step. When only a handful of markers is needed to identify the cell type of interest, indirect detection with unlabeled primary antibodies and fluorophore-labeled secondary antibodies is often preferred. One reason for this is that indirect detection offers increased flexibility for panel design due to the wide range of secondary antibodies that are commercially available. In addition, indirect detection provides signal amplification as a result of multiple secondary antibodies binding to each primary antibody, which can be especially useful when studying low density antigens. When selecting secondary antibodies for flow cytometry, there are several key factors to consider. First, the excitation and emission maxima of each fluorophore should be compatible with the flow cytometer’s lasers and detectors, respectively. Second, because the signal from any fluorophore used in a multicolor flow cytometry panel can spill over into other detectors than that assigned for its measurement, it is important to choose fluorophores that are spectrally distinct. Other general recommendations include considering the use of isotype- or subclass-specific secondary antibodies for greater versatility in reagent selection; choosing secondary antibodies labeled with bright fluorophores such as R-phycoerythrin (RPE) secondaries or Allophycocyanin (APC) secondaries for detecting scarce targets; and introducing tandem dyes into staining panels to increase the level of plex if the number of lasers becomes a limiting factor. It is also important to note that flow cytometry experiments based on indirect detection should always include secondary antibody only controls. These consist of samples that have been incubated only with the secondary antibody (no primary antibody incubation step) and are essential to identify any non-specific secondary antibody binding that could lead to data being incorrectly interpreted.