Protocol to evaluate virus-specific CD4⁺ and CD8⁺ T cell responses by spectral flow cytometry

T cell responses play an important role in viral clearance and prevention of infection. Here, we present a protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses using spectral flow cytometry. We describe steps for peripheral blood mononuclear cell (PBMC) thawing and stimulation with SARS-CoV-2 spike peptide pools. We then describe detailed procedures for cell confining, staining, and fixation. This protocol has potential application in spectral flow cytometry on other virus-specific T cell responses. For complete details on the use and execution of this protocol, please refer to Zhao et al.¹.