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Updated: Jul 17, 2026
Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Published on: January 7, 2019
Protocol for deep phenotyping of human antigen-specific T cells across conventional and unconventional subsets by
Jiahui Du1, Mingzhe Zeng1, Ziqian He1
1National Key Laboratory of Immunity and Inflammation, Institute of Immunology, Naval Medical University, Shanghai, China.
Abstract:
Analyzing antigen-specific T cells at the subset level is crucial for studying antiviral immunity and vaccine efficacy. We present a 22-color spectral flow cytometry protocol to characterize these cells from human PBMCs, covering conventional αβT and unconventional (MAIT, γδ T) subsets. We describe steps for PBMC thawing and plate setup, peptide stimulation, three-step surface staining, and intracellular staining. We then detail procedures for sample acquisition and Flowjo data analysis. This approach enables precise immunophenotyping with limited samples.
