A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8⁺, CD4⁺), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells. Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.