Background The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology detects discrete signals from antibodies tagged with isotopically enriched metals, facilitating highly multiparametric characterization on a single-cell level. CyTOF enables analysis of over 50 markers simultaneously without the impediments of spectral overlap or autofluorescence, allowing for reliable evaluation of immune responses. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down 30-marker antibody cocktail for single-tube staining and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations.

Methods The complex nature of the immune system requires deep interrogation at the single-cell level. Flow cytometry using CyTOF® technology detects discrete signals from antibodies tagged with isotopically enriched metals, facilitating highly multiparametric characterization on a single-cell level. CyTOF enables analysis of over 50 markers simultaneously without the impediments of spectral overlap or autofluorescence, allowing for reliable evaluation of immune responses. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down 30-marker antibody cocktail for single-tube staining and acquisition on CyTOF systems. Using the assay and Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations.

Results The simultaneous detection of key surface and intracellular markers in this expanded panel enabled comprehensive analysis of immune cell activation and antigen-specific recall responses in stimulated PBMC. The functional landscape of activated immune cells was mapped using the Cen-se′™ dimensionality reduction tool, which showed T cell populations that co-expressed activation markers including IL-2, TNFα, IFNγ, and CD69. Automatic Maxpar Pathsetter analysis revealed greater frequencies of activation markers and cytokine production in antigen-specific CD4+ and CD8+ T cells compared with global CD4+ and CD8+ T cell populations across all donors tested. Importantly, the polyfunctionality detected in these subsets is a correlate of T cell efficacy.

Conclusions Taken together, this 47-marker panel and automated analysis unlocked deeper immunophenotyping and functional profiling of activated immune cells, providing insight into the cell-mediated adaptive immune response to foreign targets. Such phenomena are hallmarks for research on infection, vaccine development, and cancer immunotherapy.

For Research Use Only Not for use in diagnostic procedures.