Flow cytometry utilises fluorescently‑labelled antibodies to measure protein expression on single cells. The high sensitivity and high-throughput nature of the technique make it suitable for the characterisation of immune cell subsets. However, spectral overlap limits the number of fluorophores that can be used simultaneously and increasingly detailed characterisation of subsets requires access to more of the light spectrum. Recently, spectral cytometry, which measures the whole emission spectrum of each fluorescent signal, rather than measuring target regions of the emitted spectrum, has been presented as an alternative to standard flow cytometry. This technique uses the whole signature of a fluorophore, rather than the peak emission, to un-mix all fluorescent signals in a sample, allowing more fluorophores of similar emission wavelengths to be used simultaneously. As spectral cytometry exhibits potential advantages in single-cell measurement, we compared the performance of a spectral cytometer with conventional cytometry in various ways.
We labelled compensation beads and Qdots with a wide range of fluorophore‑conjugated antibodies to determine the ability of spectral cytometry to separate multiple overlapping fluorophores at different emission intensities. Next, we stained cells from homeostatic and infected murine brain, lymph nodes and bone marrow with panels of 12-16 antibodies, before acquiring samples in parallel on a 3-laser spectral cytometer (Cytek Aurora) and a conventional high-parameter cytometer (BD custom LSR-II). We will present data on overall panel performance, resolution of dim and rare populations and intracellular staining, as well as separation of signal from background. We also assessed signal resolution on inherently highly auto fluorescent brain cells following subtraction of autofluorescence on the spectral cytometer. While spectral cytometry clearly enables the use of more fluorophores in a single assay, here we provide a specific analysis of the applied comparative performance of conventional and spectral cytometers in a variety of experimental uses.