High-parameter flow cytometry by CyTOF empowers comprehensive immune profiling of splenic and pulmonary tissues in aged mice

Abstract Flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infection, and senescence. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased immune-profiling approach and maximizes insights from precious samples. To study the immune system in aged (75 weeks old) mice, a mouse immune profiling panel (32 antibodies) was selected from Standard BioTools™ catalogs to detect key lineage and functional surface markers. Maxpar® Pathsetter™ software was used to create analytical models for automated high-dimensional analysis. In mouse spleen, the panel identified 38 immune subsets (T cells, B cells, NK cells, dendritic cells, neutrophils, etc.) and enabled high-fidelity detection of over 13 functional markers mediating proliferation, activation, inhibition, migration, and cell adhesion. With 10 additional Maxpar antibodies, the 42-antibody panel characterized 55 immune subsets in aged lungs. Many aging-associated immune alterations were identified, such as PD1+ T cell enrichment and NK cell maturation defects. CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as alveolar macrophages (AMs). The pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged mice. Thus, utilizing mouse immune profiling panels and the automated analytical tools offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate mechanistic studies in mice and expand the understanding of human immunity. Flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infection, and senescence. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased immune-profiling approach and maximizes insights from precious samples. To study the immune system in aged (75 weeks old) mice, a mouse immune profiling panel (32 antibodies) was selected from Standard BioTools™ catalogs to detect key lineage and functional surface markers. Maxpar® Pathsetter™ software was used to create analytical models for automated high-dimensional analysis. In mouse spleen, the panel identified 38 immune subsets (T cells, B cells, NK cells, dendritic cells, neutrophils, etc.) and enabled high-fidelity detection of over 13 functional markers mediating proliferation, activation, inhibition, migration, and cell adhesion. With 10 additional Maxpar antibodies, the 42-antibody panel characterized 55 immune subsets in aged lungs. Many aging-associated immune alterations were identified, such as PD1+ T cell enrichment and NK cell maturation defects. CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as alveolar macrophages (AMs). The pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged mice. Thus, utilizing mouse immune profiling panels and the automated analytical tools offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate mechanistic studies in mice and expand the understanding of human immunity.