Capturing the full spectrum of T cell responses with spectral flow cytometry.

Over a decade has passed since the first commercial spectral flow cytometry (SFC) instrument was introduced. Unlike conventional flow cytometers, SFC utilizes an array of detectors to capture the full emission spectrum of fluorochromes, from which composite signatures are deconvoluted using an unmixing algorithm. This allows fluorochromes with overlapping peaks to be used within the same panel, enabling panels with up to 50 parameters. As its availability increases, more immunologists are looking to incorporate SFC into their experiments. One area of research benefiting from the larger SFC panels is the characterization of rare cells, including antigen-specific T cells identified directly ex vivo using either antigen stimulation or major histocompatibility complex-peptide multimers. In this brief review, we outline some practical considerations when combining ex-vivo T cell stimulation with SFC, drawing on our transition from conventional to SFC. Key aspects include designing the experiment and panel for stimulated cells, acquiring high-quality reference controls, strategies to manage autofluorescence and an overview of the data analysis, including both manual and computational approaches.