A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

Abstract

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

Inference of immune cell composition on the expression profiles of mouse tissue

“…Based on their cell surface markers, immune cell types could be qualitatively and quantitatively measured via several experimental methods, including flow cytometry7, affinity purification8, and immunohistochemistry9. Using flow cytometry, Gunn et al .…”

“…Using flow cytometry, Gunn et al . developed a protocol that could accurately quantify 11 immune cell types in non-lymphoid tissues7. McGuire et al 9.…”

Temporally Distinct PD-L1 Expression by Tumor and Host Cells Contributes to Immune Escape

“…Antibodies used for comprehensive flow cytometry analysis of cell subsets in tumors are listed in Supplementary Table S1 (31). Additionally, the following monoclonal antibodies (mAbs) were used and purchased from BD Bioscience, anti-CD16/CD32 (2.4G2), PE-conjugated anti-SiglecF (E50-2440), and anti–H2-K b (AF6-88.5).…”

Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire

“…For analysis of MAIT cells, spleens and lungs were collected from 8–10 week old mice. Single-cell suspensions from lung were prepared as described (Yu et al, 2016), with modifications. Briefly, mice were euthanized via CO 2 asphyxiation and perfused with 25 ml phosphate-buffered saline (PBS) through the right atrium to flush erythrocytes and non-adherent leukocytes from the pulmonary vasculature.…”