Characterisation of lung macrophage subpopulations in copd. Macrophage activation markers view the complex biology of macrophages poster showing the expression levels of different macrophage markers following development, macrophages encounter diverse stimuli, which can alter their transcriptional. This was further confirmed by examining the wlc macrophages for m1 phenotype markers. Using a mouse model, we discovered a kind of lung wbc that survives chemotherapy. Analysis of single and doublestained alveolar macrophages. We validated this panel using normal lung tissue and tissue from patients with chronic obstructive pulmonary disease and lung fibrosis. A portion of these cells expressed cd1a 22% and my4 60%. Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages, and monocytes in the human lung and in bronchoalveolar lavage fluid. Cd163 and ccr7 as markers for macrophage polarization in.
Flow cytometry reveals similarities between lung macrophages in. It allows multiparameter analysis of cell populations based on the expression of specific cell surface andor intracellular molecules. In copd, these macrophages are out of control and produce huge amounts of enzymes that break down lung tissue and more proteins that cause inflammation. Flow cytometrybased analysis and sorting of lung dendritic cells 241 cd68 van haarst et al. These cells were subjected to analysis by flow cytometry to characterize the expression of activation markers, chemoattractant receptors, and surface markers characteristic of either m1 or m2type macrophages. Mouse lung and spleen natural killer cells have phenotypic. They evaluated a macrophage population in balf and in lung tissue using multicolour cytometry and confocal microscopy. Pdf the lung hosts multiple populations of macrophages and dendritic cells, which play a. The high autofluorescent cell fraction represented alveolar macrophages which were strongly positive for aph, hladr, cd68, rfd7, and rfd9. A protocol for the comprehensive flow cytometric analysis. Analysis of microglia and monocytederived macrophages. The essential feature of the method is the classification of the bal cells during data analysis into subpopulations having similar autofluorescence on. We developed a standardized protocol to process the lungs for flow cytometric analysis see online supplement for details.
However, the expression levels of mertk and cd169 are significantly lower than that of cd64, making them less useful as a primary means to identify macrophages via flow cytometry. The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Antigen expression was quantified by flow cytometry and expressed as relative. Dysregulated functions of lung macrophage populations in copd. Because this panel recognises surface markers, it can be used for sorting live cells for subsequent gene expression, epigenetic or other analysis. Recently, a 5 and a half year study showed that some smokers that can breathe normally have. Exogenous remodeling of lung resident macrophages protects. Jun 15, 2018 blood monocytes were isolated from normal nonsmokers 14, current smokers, patients with moderate 9, and severe copd 11. Identification of myeloid cell subsets in murine lungs using flow. Flow cytometrybased analysis and sorting of lung dendritic cells. Two hundred microliters wholeblood or 106 bal cells were stained for flow cytometry as previously described to analyze the expression of sur face markers and.
Flow cytometry applications in the study of immunological. Identification of myeloid cell subsets in the lungs by facs. Monocytes and macrophages are phagocytes, acting in both nonspecific defence or innate immunity as well as specific defense or cellmediated immunity of vertebrate animals. Paired t tests were used to compare flow cytometry cell surface marker expression and gene expression data between small and large macrophages, and between interstitial and alveolar macrophages. We developed a protocol for the flow cytometric analysis of nonlymphoid tissues, including methods of tissue preparation, a 10fluorochrome panel for cell staining, and. Flow cytometry and highcontent imaging to identify markers of monocytemacrophage differentiation dev mittar, rosanto paramban, and catherine mcintyre bd biosciences contents 1 abstract 2 introduction 3 for the expression of 242 human cell surface proteins by flow cytometry or objective 5 methods 9 results 17 conclusions 18 references abstract. Mouse anti human macrophages, clone mac387 recognizes the l1 or calprotectin molecule, an intracytoplasmic antigen comprised of a 12 kda alpha chain and a 14 kda beta chain. Flow cytometry characterization of circulating cells belonging to the monocytemacrophage lineage was performed using specific m1 cd80, cd86, tlr2 and tlr4 and m2 surface markers cd204, cd163 and cd206. Innate lymphoid cell gating strategies for flow cytometry flow cytometry is essential for the study of innate lymphoid cells ilcs. Pdf flow cytometric analysis of macrophages and dendritic.
Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycininduced lung injury, whereas. Macrophage subpopulation cellsurface expression of immunological markers and phagocytic ability were assessed by flow cytometry. We developed a 10fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. Alveolar macrophages ams harvested from 32 hivinfected patients with respiratory problems opportunistic pulmonary infections, n12. Human alveolar macrophages predominately express combined. Pulmonary monocytes and macrophages are believed to function in a wide range of biological roles, including host defense against foreign organisms, maintenance of immunological homeostasis in the lung, presentation of antigen to lymphocytes, and migration to sites of tissue injury and inflammation. Flow cytometry can be used for specific assessments, such as to identify different tcell phenotypes 7, 31.
Flow cytometry reveals similarities between lung macrophages. Flow cytometry is a mainstream tool for studying immune system, however, a systematic flow cytometric approach to accurately and consistently identify subsets of macrophages and dendritic cells. Flow cytometric analysis of macrophages and dendritic cell. Blood monocytes were isolated from normal nonsmokers 14, current smokers, patients with moderate 9, and severe copd 11. A protocol for the comprehensive flow cytometric analysis of. Tumor associated macrophages support the growth of fgf9. American journal of respiratory cell and molecular biology. Although originally described as binding to epitopes common to both the alpha and beta chains flavell et al. Interstitial macrophages, together with pulmonary monocytes and dendritic. Analysis of microglia and monocytederived macrophages from.
Expression of surface markers on alveolar macrophages from. Specifically, this panel identifies alveolar macrophages, interstitial macrophages, ly6chi classical. Mouse anti pig cd163 antibody, clone 2a1011 recognises porcine cd163, a 120 kda single pass type 1 transmembrane cell surface glycoprotein expressed on cells of the monocytemacrophage lineage. We developed a 10fluorochrome flow cytometry panel for the identification and. Flow cytometry and highcontent imaging to identify markers. A new therapeutic pathway in fibrosing lung disease. Flow cytometric analysis of the macrophages and dendritic. I would like to analyse lung macrophage by flow cytometry and wonder what to choose between cd64 and f4f80 antibody. Differential expression of surface markers classifies macrophages to either. In contrast, spleen is a lymphatic organ connected to the blood. Is f4f80 a good marker for murine lung macrophages. Contour plots and gating strategy used for the identification of major immune cell populations plus mast cells and basophils in normal mouse lungs. These findings were confirmed using a second model of acute lung inflammation generated by. We report here a method which, by overcoming the problem of autofluorescence, makes possible for the first time two color analysis of surface markers on alveolar macrophages by flow cytometry.
Cd68 is used primarily in histological staining to identify macrophages, but has been used for this purpose in some flow cytometric studies. Innate lymphoid cell gating strategies for flow cytometry. This antibody was used to label alveolar macrophages from mouse lung digested tissue. Flow cytometry has been used in the determination of surface markers on eosinophils10 and neutrophils in asthmatic patients. The expression levels of cd163 vary during the course of macrophage differentiation. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells dcs accurately and consistently in the normalmouse lung has not been described. Flow cytometry and highcontent imaging to identify.
Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. Flow cytometry analysis of alveolar macrophages after surfactant treatment with 120 mgkg. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. American journal of respiratory cell and molecular biology, 541, pp. Phagocytosis was determined by flow cytometry evaluation of fitclabeled dextran uptake. Identification of myeloid cell subsets in murine lungs. Jul, 2015 unfortunately, no available surface marker is well correlated with any other marker, at either the protein or mrna level hume, 2008, 2012. Activation and polarization of circulating monocytes in. Flow cytometry is a mainstream tool for studying immune system, however, a systematic flow cytometric approach to accurately and consistently identify subsets of macrophages and dendritic cells dcs in the normal mouse lung has not been described.
However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. The heterogeneity of lung macrophages in the susceptibility. This work was supported by a pulmonary hypertension. Peripheral blood monocytes, on the other hand, can be subdivided into two. Accurate and simple discrimination of mouse pulmonary. Although species and strain dependent, in the fischer rat, the most common cell types include. Flow cytometrybased immunophenotyping was used to characterise am.
Alveolar macrophages are specialised resident phagocytes in the alveolus, constituting the first line of immune cellular defence in the lung. Prevention of infection by vaccination also requires wbcs, and thus has not been effective in saving patients with low wbc counts during chemotherapy. Supplementary figure 1 phenotype and geneexpression profiling of arterial macrophages. Using flow cytometry, we identified the cells responsible for uptake of cyanine 5conjugated folate as fr. We examined these two functions in facssorted ims, ams, and lung dcs. As the lung microenvironment is challenged and remodelled by inhaled pathogens and air particles, so is the alveolar macrophage pool altered by signals that maintain andor replace its composition. This antibody was used to label alveolar macrophages from. Exogenous surfactant changes the phenotype of alveolar. Chronic obstructive pulmonary disease copd is a diverse respiratory disease characterised by bronchiolitis, small airway obstruction, and emphysema. Flow cytometric analysis of the macrophages and dendritic cell subsets in the. Siglecf flow cytometry antibodies the general focus of my work is to analyze the response different cell types in mouse lungs after pulmonary bacteria infection. Which marker is best for human macrophages in flow cytometry.
Unfortunately, no available surface marker is well correlated with any other marker, at either the protein or mrna level hume, 2008, 2012. Macrophages, unlike dcs, have a high capacity to phagocytose but are poor inducers of t cell proliferation 21, 22. Feb 25, 20 the lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. This experiment showed that changes in the expression of cell surface markers can increase on lipidladen alveolar macrophages and that the change can be detected by flow cytometry. Singlecell suspensions freshly obtained from collagenasedigested lung tissue were stained with a. Flow cytometric analysis of blood monocytes and alveolar. Alveolar macrophages expressed markers of alternatively activated macrophages during both. Alveolar macrophages expressed markers of alternatively activated. Immune surveillance of the lung by migrating tissue. We developed a protocol for the flow cytometric analysis of nonlymphoid tissues, including methods of tissue preparation, a 10fluorochrome panel for cell staining, and a standardized gating. Antigen expression was quantified by flow cytometry and expressed as relative linear median. This protocol describes the procedures for isolating the mononuclear. Flow cytometry is a mainstream tool for studying the immune system.
Misharin av1, moralesnebreda l, mutlu gm, budinger gr, perlman h. Cytometry protocol for macrophage isolation from mice lungs. Flow cytometry reveals similarities between lung macrophages in humans and mice. The lung is an organ in contact with respiratory pathogens and the site of inflammatory disorders triggered by the respiratory environment. Preparation of rat lung cells for flow cytometry springerlink. Flow cytometric analysis of mouse lungs using extended antibody panel to identify mast cells and basophils. We want to see if we can find these macrophages in the lungs of smokers before they get copd. The highest levels of cd163 expression are found on tissue.
Nk cells are lymphocytes of the innate immune system which are a first line of defense against infections and tumor cells, in bone marrow and peripheral organs like lung and spleen. Anti human macrophages antibody, clone mac387 biorad. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells dcs accurately and consistently in the normal mouse lung has not been described. Pdf flow cytometric analysis of macrophages and dendritic cell. Cytometry protocol for macrophage isolation from mice lungs dear flowers, this is an unusual request. Siglecf staining of alveolar macrophages in mouse lungs. Cytometry protocol for macrophage isolation from mice lungs dear ami, unless you. Jci lung interstitial macrophages alter dendritic cell. Analysis of single and doublestained alveolar macrophages by. Implications for understanding interstitial lung macrophages in. In contrast, spleen is a lymphatic organ connected to the blood system. Infectious complications can be lethal in patients with cancer when chemotherapy depletes white blood cells wbcs needed to clear microbes.
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