Using flow cytometry, we identified the cells responsible for uptake of cyanine 5conjugated folate as fr. We validated this panel using normal lung tissue and tissue from patients with chronic obstructive pulmonary disease and lung fibrosis. I would like to analyse lung macrophage by flow cytometry and wonder what to choose between cd64 and f4f80 antibody. 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. Analysis of microglia and monocytederived macrophages from. Flow cytometrybased analysis and sorting of lung dendritic cells. This protocol describes the procedures for isolating the mononuclear. 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. 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.
Cd163 and ccr7 as markers for macrophage polarization in. Recently, a 5 and a half year study showed that some smokers that can breathe normally have. 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. Analysis of single and doublestained alveolar macrophages. A protocol for the comprehensive flow cytometric analysis. Innate lymphoid cell gating strategies for flow cytometry flow cytometry is essential for the study of innate lymphoid cells ilcs. Alveolar macrophages ams harvested from 32 hivinfected patients with respiratory problems opportunistic pulmonary infections, n12. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. This work was supported by a pulmonary hypertension. Flow cytometry can be used for specific assessments, such as to identify different tcell phenotypes 7, 31. Jul, 2015 unfortunately, no available surface marker is well correlated with any other marker, at either the protein or mrna level hume, 2008, 2012. 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.
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 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. Flow cytometry applications in the study of immunological. 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. In contrast, spleen is a lymphatic organ connected to the blood. Identification of myeloid cell subsets in murine lungs using flow. Flow cytometrybased analysis and sorting of lung dendritic cells 241 cd68 van haarst et al. Characterisation of lung macrophage subpopulations in copd. Flow cytometry has been used in the determination of surface markers on eosinophils10 and neutrophils in asthmatic patients.
Monocytes and macrophages are phagocytes, acting in both nonspecific defence or innate immunity as well as specific defense or cellmediated immunity of vertebrate animals. A new therapeutic pathway in fibrosing lung disease. Flow cytometry reveals similarities between lung 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. The high autofluorescent cell fraction represented alveolar macrophages which were strongly positive for aph, hladr, cd68, rfd7, and rfd9. Antigen expression was quantified by flow cytometry and expressed as relative. Dysregulated functions of lung macrophage populations in copd. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. Pdf flow cytometric analysis of macrophages and dendritic cell. 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.
The lung is an organ in contact with respiratory pathogens and the site of inflammatory disorders triggered by the respiratory environment. Unfortunately, no available surface marker is well correlated with any other marker, at either the protein or mrna level hume, 2008, 2012. A portion of these cells expressed cd1a 22% and my4 60%. Flow cytometry reveals similarities between lung macrophages in. This was further confirmed by examining the wlc macrophages for m1 phenotype markers. Flow cytometric analysis of macrophages and dendritic cell. It allows multiparameter analysis of cell populations based on the expression of specific cell surface andor intracellular molecules. Analysis of microglia and monocytederived macrophages. Pdf flow cytometric analysis of macrophages and dendritic. Anti human macrophages antibody, clone mac387 biorad. 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.
The expression levels of cd163 vary during the course of macrophage differentiation. Pdf the lung hosts multiple populations of macrophages and dendritic cells, which play a. Identification of myeloid cell subsets in murine lungs. Flow cytometry analysis of alveolar macrophages after surfactant treatment with 120 mgkg. Differential expression of surface markers classifies macrophages to either. Although the antibodybased recognition of cellsurface markers has been widely. We developed a 10fluorochrome flow cytometry panel for the identification and. Alveolar macrophages are specialised resident phagocytes in the alveolus, constituting the first line of immune cellular defence in the lung. American journal of respiratory cell and molecular biology, 541, pp. This antibody was used to label alveolar macrophages from mouse lung digested tissue. Prevention of infection by vaccination also requires wbcs, and thus has not been effective in saving patients with low wbc counts during chemotherapy. 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.
Is f4f80 a good marker for murine lung macrophages. Which marker is best for human macrophages in flow cytometry. 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. Implications for understanding interstitial lung macrophages in. Because this panel recognises surface markers, it can be used for sorting live cells for subsequent gene expression, epigenetic or other analysis. 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. Immune surveillance of the lung by migrating tissue. They evaluated a macrophage population in balf and in lung tissue using multicolour cytometry and confocal microscopy. Analysis of single and doublestained alveolar macrophages by. American journal of respiratory cell and molecular biology. Tumor associated macrophages support the growth of fgf9. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycininduced lung injury, whereas.
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 cytometrybased immunophenotyping was used to characterise am. Antigen expression was quantified by flow cytometry and expressed as relative linear median. 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. Innate lymphoid cell gating strategies for 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. Chronic obstructive pulmonary disease copd is a diverse respiratory disease characterised by bronchiolitis, small airway obstruction, and emphysema. Expression of surface markers on alveolar macrophages from. 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.
Macrophages, unlike dcs, have a high capacity to phagocytose but are poor inducers of t cell proliferation 21, 22. Specifically, this panel identifies alveolar macrophages, interstitial macrophages, ly6chi classical. In addition to the identification of the different infiltrating cell populations, the noncellular composition of the lung can be investigated using bal. Flow cytometry reveals similarities between lung macrophages in humans and mice.
Exogenous remodeling of lung resident macrophages protects. Activation and polarization of circulating monocytes in. Cytometry protocol for macrophage isolation from mice lungs dear ami, unless you. In contrast, spleen is a lymphatic organ connected to the blood system. Flow cytometry and highcontent imaging to identify. The essential feature of the method is the classification of the bal cells during data analysis into subpopulations having similar autofluorescence on. Jci lung interstitial macrophages alter dendritic cell. Although species and strain dependent, in the fischer rat, the most common cell types include.
These findings were confirmed using a second model of acute lung inflammation generated by. Macrophage subpopulation cellsurface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Flow cytometric analysis of mouse lungs using extended antibody panel to identify mast cells and basophils. Feb 25, 20 the lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Cd68 is used primarily in histological staining to identify macrophages, but has been used for this purpose in some flow cytometric studies. Phagocytosis was determined by flow cytometry evaluation of fitclabeled dextran uptake. 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. We developed a standardized protocol to process the lungs for flow cytometric analysis see online supplement for details. The highest levels of cd163 expression are found on tissue. Alveolar macrophages expressed markers of alternatively activated macrophages during both. Preparation of rat lung cells for flow cytometry springerlink. Human alveolar macrophages predominately express combined. Supplementary figure 1 phenotype and geneexpression profiling of arterial macrophages.
This antibody was used to label alveolar macrophages from. The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Flow cytometric analysis of blood monocytes and alveolar. 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.
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. Identification of myeloid cell subsets in the lungs by facs. 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. Flow cytometry is a mainstream tool for studying the immune system. 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. Interstitial macrophages, together with pulmonary monocytes and dendritic. Exogenous surfactant changes the phenotype of alveolar.
The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Using a mouse model, we discovered a kind of lung wbc that survives chemotherapy. We want to see if we can find these macrophages in the lungs of smokers before they get copd. Flow cytometric analysis of the macrophages and dendritic cell subsets in the. Dear ami, typo on my side, should be collagenasednaase, not tryps.
A protocol for the comprehensive flow cytometric analysis of. 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. Flow cytometric analysis of the macrophages and dendritic. The heterogeneity of lung macrophages in the susceptibility. Mouse lung and spleen natural killer cells have phenotypic. Peripheral blood monocytes, on the other hand, can be subdivided into two. Alveolar macrophages expressed markers of alternatively activated. Jun 15, 2018 blood monocytes were isolated from normal nonsmokers 14, current smokers, patients with moderate 9, and severe copd 11. Blood monocytes were isolated from normal nonsmokers 14, current smokers, patients with moderate 9, and severe copd 11. Flow cytometry and highcontent imaging to identify markers. Contour plots and gating strategy used for the identification of major immune cell populations plus mast cells and basophils in normal mouse lungs. Although originally described as binding to epitopes common to both the alpha and beta chains flavell et al. Siglecf staining of alveolar macrophages in mouse lungs.
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