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.

Fig 1. Flow cytometric analysis of mouse lung.

A. Contour plots of windows and gating strategy used for the identification of major immune cell populations in normal mouse lungs. Gates containing multiple cell populations are numbered (R1-R9). Gates containing a single cell population are labeled with the included cell type. These include: T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes (iMono), resident monocytes (rMono), alveolar macrophages (AMФ), interstitial macrophages (iMФ), CD11b^- dendritic cells (CD11b^- DC), and CD11b⁺ dendritic cells (CD11b⁺ DC). The distribution of cells within all panels is representative of 20 independent experiments. B. Photomicrographs of Jenner-Giemsa stained individual mononuclear phagocyte cell types purified by FACS using the gating strategy shown in A.

Fig 2. Flow cytometric analysis of other non-lymphoid organs.

A. Dot plots showing selected windows and gating strategy as applied to the identification of major immune cell populations in the indicated tissues. The gating strategy begins on the top row, which is gated on R5 from Fig 1. Selected populations specifically identified and color-coded include monocytes (blue), macrophages (MФ, green), and dendritic cells (DC, orange). Populations not labeled conform to those shown in Fig 1. Figures are representative of 3 independent experiments. B. Contour plots of CD11c vs. MHC class II expression for monocytes (blue), macrophages (green), and dendritic cells (orange) from gates shown in panel A. C. Pie charts showing the relative frequencies of all major immune cell types in the indicated tissues. The upper small charts display cell type frequencies as a percentage of total tissue of CD45⁺ cells. The lower large charts display cell type frequencies as a percentage of total tissue of myeloid cells. Percentage represents the means of values obtained in 3 independent experiments.

Fig 3. Expression of common myeloid markers on specific cell populations in non-lymphoid tissues.

A. Histograms displaying the expression levels of MerTK, CD169, F4/80, CD14, CD68, and CD206 on macrophages (MФ), dendritic cells (DC), and monocytes in the indicted tissues. Filled grey plots represent isotype controls. All panels are representative of 3 independent experiments. B. Contour plots of CD11b vs. Green Fluorescent Protein expression in macrophages (upper row) and dendritic cells (lower row) in the indicated tissues. Each panel shows cells obtained from wild type mice (black) overlaid with cells obtained from Cx3cr1^wt/GFP knock-in mice (green). All panels are representative of 3 independent experiments.

Fig 4. Flow cytometric analysis of H1N1 influenza infected lung tissues.

A. Dot plots showing gating strategy used to identify monocytes and macrophage (MФ) subpopulations in H1N1 influenza infected lung tissues. The gating strategy begins on the top row, which is gated on R5 from Fig 1. B. Histograms depicting CD169, MerTK, CD14, F4/80, CD206, and CCR2 expression on interstitial macrophages (IMФ), exudative macrophages (ExMФ), alveolar macrophages (AMФ), and Ly6C^high inflammatory monocytes (iMono). C. Line graph depicting immune cell profiles on day 0, 3, and 7 after H1N1 influenza exposure. And, n = 3 for day 0; n = 9 for day 3; and n = 7 for day 7. *p<0.05, **p<0.01, and ***p<0.001, compared to day 0. #p<0.05, ##p<0.01, and ###p<0.001, compared to day 3.

Fig 5. Flow cytometric analysis of LPS exposed lung tissues.

A. Dot plots showing gating strategy used to identify monocytes and macrophage (MФ) subpopulations in intranasal LPS exposed lung tissues. The gating strategy begins on the top row, which is gated on R5 from Fig 1. B. Histograms depicting CD169, MerTK, CD14, F4/80, CD206, and CCR2 expression on interstitial macrophages (IMФ), exudative macrophages (ExMФ), alveolar macrophages (AMФ), and Ly6C^high inflammatory monocytes (iMono). C. Line graphs depicting immune cell profiles on 0, 1, 2, 3, and 4 days after LPS exposure. And, n = 3 for day 0; n = 6 for day 1 and 4; n = 5 for day 2 and 3. *p<0.05, **p<0.01, and ***p<0.001, compared to day 0.

Fig 6. Flow cytometric analysis of primary mammary tumors.

A. Dot plots showing gating strategy used to identify macrophages (MФ) and inflammatory monocytes in normal mammary tissues and MMTV-PyMT, Met-1, and E0771 mammary tumors. The gating strategy begins on the top row, which is gated on R5 from Fig 1. B. Pie charts showing the relative frequencies of major immune cell types in normal mammary tissues and mammary tumors formatted as in Fig 2C. n = 2 for MMTV-PyMT tumors, and n = 3 for all other tissues. C. Dot plots of CD11b vs. MHC class II and CD11b vs. Ly6C expression for normal tissue and tumor-associated macrophages. All panels gated on the MФ gate in panel A. D. Histograms of MerTK, Tie2, and CCR2 expression on the normal tissue and tumor-associated macrophage subpopulations identified in panel C. Filled grey plots represent isotype controls. All panels are representative of 3 independent experiments.

Fig 7. Comparison of myeloid cell accumulation and tumor associated macrophage phenotypes in model E0771 primary tumors and metastases.

A. Time course of neutrophil, monocyte, and macrophage accumulation in model E0771 primary tumors and metastases over a 3-week time course. n = 3, ***p<0.001 (compared to 0 weeks). ###p<0.001 (compared to 1.5 weeks). B. Dot plots of CD11b vs. MHC class II and CD11b vs. Ly6C expression on tumor-associated macrophages in model E0771 primary tumors and metastases. C. Histograms of MerTK and CCR2 expression on the tumor-associated macrophage subpopulations identified in panel B. Filled grey plots represent isotype controls. All panels are representative of 3 independent experiments.