Protocol for multi-organ isolation of murine leukocytes for flow cytometry

The immune system comprises several specialized cells across multiple organs. Here, we present a protocol for the isolation of leukocytes from murine blood, spleen, mesenteric lymph nodes, liver, kidney, and skeletal muscle. We describe steps for cell isolation and gradient density separation and provide four antibody panels to identify adaptive immune cells (e.g., CD4 + and CD8 + T cell subsets) by flow cytometry. This protocol provides a basic framework for phenotyping immune cells from in vivo mouse models. • A streamlined protocol for murine leukocyte isolation from multiple organs • Guidelines for multi-organ processing for in vivo mouse models • Instructions for multiparameter flow cytometry analyses of immune T lymphocyte subsets Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The immune system comprises several specialized cells across multiple organs. Here, we present a protocol for the isolation of leukocytes from murine blood, spleen, mesenteric lymph nodes, liver, kidney, and skeletal muscle. We describe steps for cell isolation and gradient density separation and provide four antibody panels to identify adaptive immune cells (e.g., CD4 + and CD8 + T cell subsets) by flow cytometry. This protocol provides a basic framework for phenotyping immune cells from in vivo mouse models.