Analysis of monocytes from mouse spleen by flow cytometry. Flow cytometry stainings on nonautofluorescent cd163 int cd14 high spleen monocytes, cd14 high blood monocytes gated on fscssc patterns, autofluorescent cd163 high cd14 low red pulp. In this study, the features of murine macrophages from bone marrow, spleen and. B expression of surface markers on rpm identified as shown in a. Simple propagation method for resident macrophages by coculture. Because a difference in cd11c expression intensity between dc and macrophages was noted by flow cytometry figure 1, spleen sections were stained for cd11c to investigate the. A main function of splenic red pulp macrophages is the degradation of. Cx3cr1 is a known marker of ly6cloresident monocytes 7, 25, 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. Identification and isolation of splenic tissueresident. Immune cells in the spleen and brain were measured using flow cytometry. Flow cytometry is a mainstream tool for studying the immune system. Carter, and padmakumar narayanan cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry.
Flow cytometry is a valuable technique for leukocyte analysis, but a. Our new page lists the hallmark markers typically used to phenotype several popular immune cells in research. Analysis of nk cells from mouse spleen by flow cytometry usa. Peripheral blood mononuclear cell pbmc isolation and red. Several tissues including the spleen, lung, central nervous system, liver, skin. Each macrophage subpopulation in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry. In mice, m1 macrophages can be characterized by expression of inos and m2 macrophages by arginase1 production. 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. In comparison to flow cytometric cell sorting, purity of cell isolates is usually. After 2day polarization of mature m0 macrophages with m1 and m2 stimuli, we investigated the expression of a broad panel of surface markers by flow cytometry table e1. For downstream flow cytometric analysis of nk cells, we have designed a validated multicolor flow cytometry panel, using our reafinity recombinant antibodies and viobility fixable dyes. However, differences among macrophages from these particular sources remain unclear. Aug 12, 2019 for example, biorads f480 antibody is one of the most wellcharacterized and extensively referenced mouse macrophage markers for staining spleen sections. Flow cytometry analysis of bone marrow and spleen cells.
The staining procedure used in this chapter enables the characterization. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. What are the best markers to gate all mouse splenic macrophages. A protocol for the comprehensive flow cytometric analysis of. Flow cytometric analysis of inflammatory and resident myeloid. The identification and enumeration of dendritic cell. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. There are a large number of commonly used macrophage markers such as cd14, cd16, cd64, cd68, cd71 and ccr5. During development and throughout life, macrophages reside in many tissues of the body, contributing to both the maintenance. Upon establishment of the blood circulation, these primitive macrophages are the first. Currently, there is no standardized panel for immunophenotyping myeloid cells in mouse spleen using flow cytometry. To date, most experimental studies have been performed on. For example, biorads f480 antibody is one of the most wellcharacterized and extensively referenced mouse macrophage markers for staining spleen sections.
This webinar covers the basic components of a flow cytometer, how to interpret a dye excitationemission spectrum, how data is displayed, basic gating. Which is the best marker to sort splenic macrophages from. Flow cytometry is the measurement metry of cellular cyto properties as they are moving in a fluid stream flow, past a stationary set of detectors. Leukocytes can be obtained from whole blood and a variety of tissues, such as spleen, lymph node, bone marrow and thymus. Investigation of human spleen dendritic cell phenotype and.
Gating strategy showing the analysis of monocytes from healthy mouse spleen. Immunohistochemistry panels basic spleen immunohistochemistry panel cd8, cd20, cd3, cd34, erg, wt1 red pulp lymphoid lesions cd8, cd68, cd34, dba. First, we analyzed b cell development in the bone marrow and spleen of igmtg and iggtg mice by flow cytometry. However, these publications have either identified only one of the multiple dc subpopulations or focused on a. During development and throughout life, macrophages reside in many tissues of the body, contributing to both the maintenance of tissue homeostasis and the immune response following injury or pathogenic insult.
Markers such as cd11b, cd11c, f480, gr1, ly6c, and ly6g have long been used to. 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. Flow cytometry stainings on nonautofluorescent cd163 int cd14 high spleen monocytes, cd14 high blood monocytes gated on fscssc patterns, autofluorescent cd163 high cd14 low red pulp macrophages from spleen, and mcsf and gmcsf cultured monocytederived macrophages, a cd11a, b cd11b, c cd11c, d cd18, e cd89, f cd200r, g hladr. We want to sort macrophage population from mouse spleen. Intracellular staining with the macrophage marker cd68 identified the. Dengue virus infects macrophages and dendritic cells in a. Novel markers to delineate murine m1 and m2 macrophages.
Our monoclonal antibody tnkupj recognizes mouse cd163 and can be added to any panel aiming to characterize macrophages. Mouse tissueresident macrophage markers research areas. Flow cytometry stainings on monocytes in the spleen, blood monocytes. Flow cytometry and highcontent imaging to identify. Cd147 as a key mediator of the spleen inflammatory. However, for flow cytometry, spleen samples must be mechanically homogenized and treated with collagenase to release the red pulp macrophages.
We then analyzed the cell surface antigen expression on each enzymedissociated macrophage subpopulation by means of flow cytometry. Within the tissues where they reside, macrophages receive specific signals that determine their phenotypes and functional programs. The markers that best characterized m1 macrophages were cd64 the highaffinity fc. In addition, we provide an optimized gating strategy for the analysis of nk cells and characterization of nk cell maturation stages. Preparation of peritoneal macrophages, bone marrow. The staining procedure used in this chapter enables the characterization of mouse macrophages by a flow cytometry antibody panel which can be used for both bone marrowderived macrophages bmm and macrophages derived from other tissues, such as the mouse spleen or. Representative data shown from more than three independent experiments. The spleen is a particularly convenient source for murine immune cells. Cd141, is used for visualization of human dendritic cells. As a panmacrophage marker in facs you could use f480 or cd64, again depending on which tissue you are looking at. Because for spleen you need a much different panel than for lung.
The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Expression profiles of macrophage markers by flow cytometry. However, their phenotypical and functional characteristics remain incompletely understood due to the difficulty of identification and isolation by flow cytometry. Characterization of murine macrophages from bone marrow. Learn more about mouse tissueresident macrophage markers including related. As a result of recent intensive flow cytometric analyses, splenic. Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. Flow cytometry and fluorescenceactivated cell sorting facs. Which marker is best for human macrophages in flow cytometry. Flow cytometry workflow solutions proteome profiler antibody arrays. Therefore, human macrophages from both categories can be characterized by the profile of cytokines produced and the expression of cell surface markers.
Newly defined, key markers include the transcription factors blimp1 and xpb1s, which allow analysis of these key regulators in different bcell subsets. To optimize f480 staining by flow cytometry in mouse spleen, we. Multicolor flow cytometry allows the use of multiple. Macrophages in the spleen regulate the immune response and.
Peripheral blood mononuclear cell pbmc isolation and red cell lysis procedures introduction. Flow cytometry and highcontent imaging to identify markers of monocytemacrophage differentiation dev mittar, rosanto paramban, and catherine mcintyre bd biosciences contents 1 abstract 2. Immunophenotyping of the spleen biopsy by flow cytometry shows a t cell population with no aberrant loss or aberrant expression of t cell markers, a b cell population that is negative for cd5, cd10, no surface lightchain restriction. A protocol for the comprehensive flow cytometric analysis. How to isolate macrophages from mouse spleen and liver for downstream functional assays by facs. Flow cytometry and highcontent imaging to identify markers.
Isolation of splenic macrophage subpopulations using an enzyme cocktail. Mice were infected with 10 5 pfu denv2 d2s10 and euthanized at day 2. Based on the expression of cell surface markers, mouse monocytes can be. Flow cytometric analysis of inflammatory and resident. Identification and isolation of splenic tissueresident macrophage. Red pulp macrophages in the human spleen are a distinct. Flow cytometry analysis of undifferentiated bat bone marrow cells ad, in green, mcsf differentiated bmdms eh, in purple and gmcsf differentiated bmdms il, in blue demonstrating. A novel ly6cly6gbased strategy to analyze the mouse. Mice were infected with 10 5 pfu denv2 d2s10 and euthanized at day 2 after infection, and then spleen cells were analyzed by flow cytometry. This tnkupj antibody can be used with the below nonexclusive list of mouse markers table 1.
The accurate identification and enumeration of these subsets are. A novel ly6cly6gbased strategy to analyze the mouse splenic. What are the best markers to gate all mouse splenic macrophages during facs sorting. Which is the best marker to sort splenic macrophages from mice by. Functional assays byflow cytometry purdue university. Selecting the right antibody for flow cytometry biocompare. Peritoneal macrophages, bone marrow, thymus and spleen cells download pdf this method provides a general procedure for use with cell suspension cells acquired from the peritoneum, bone marrow, thymus and spleen. Phenotyping cells by flow cytometry is a powerful way to identify cell type and any morphological changes during cell culture. Jan 15, 2020 flow cytometry analysis of undifferentiated bat bone marrow cells ad, in green, mcsf differentiated bmdms eh, in purple and gmcsf differentiated bmdms il, in blue demonstrating. Aug 11, 2015 the spleen is a particularly convenient source for murine immune cells. Flow cytometry provides an important, reliable, and precise tool to separate and analyse different cells based on th eir specific phenotypic properties.
A group of macrophages, named dendritic cells, acquires the capacity of antigen presentation to the cognate immune system. Macrophages in the spleen regulate the immune response and immune tolerance date. Analysis of monocytes from mouse spleen by flow cytometry usa. Splenic red pulp macrophages are intrinsically superparamagnetic. Flow cytometry when normal splenic tissue is sampled for. Jul 30, 2007 macrophages in the spleen regulate the immune response and immune tolerance date. Our monoclonal antibody tnkupj recognizes mouse cd163 and. To achieve a panel for immunophenotyping splenic myeloid cells, various cell surface markers were tested by flow cytometry and fluorescenceactivated cell sorting facs analysis. However, these publications have either identified only one of the multiple dc subpopulations or focused on a specific tissue such as the lung, blood, or bone marrow, not all identifiable dc populations present in the spleen or peyers patches.
Peritoneal macrophages, bone marrow, thymus and spleen cells download pdf this method provides a general procedure for use with cell suspension cells. I have used the following markers in a flow cytometry panel to assess mouse samples spleen and liver derived leucocyte suspensions for numbers of antigenpresenting cells. Flow cytometry analysis of bone marrow and spleen cells openi. In vivo, cd38 revealed an endotoxemiainduced macrophage population.
Although the macrophage markers cd64 and mertk were similarly expressed on all the subpopulations, the expression profile of tim4, ly6c, cd11c and mhc class ii was different among these subpopulations. Human peripheral blood mononuclear cell pbmcderived dendritic cells stained with brilliant violet 510 antihuman cd11c antibody clone 3. If incorporating antimouse cd163, clone tnkupj into a flow cytometry panel, what other markers would be recommended for use and why. We further confirmed these data by flow cytometry and show that m1 and m2 macrophages can be distinguished by their relative expression of cd38 and egr2. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Compared to gr1, ly6cly6g markers were better for identifying neutrophils, eosinophils, and both subsets of monocytes macrophages in mouse spleen. Flow cytometry analysis shows less than 1% of the cells from the lung biopsy sample expressing leukocytic markers cd45. Markers such as cd11b, cd11c, f480, gr1, ly6c, and ly6g have long been used to identify various splenic cell myeloid populations. Representative flow cytometry dot plots are shown from 1 of 2 independent experiments. They showed that cd68 expression could be used to distinguish cgd from crohns disease in patients with colonic inflammation when measured in conjunction with levels of other cd markers 5.
Flow cytometry phenotyping of bone marrowderived macrophages. There are very few unique macrophage markers and often a number of markers will be required to identify your cell type. A cd38egr2based flow cytometry assay was capable of distinguishing m1 and m2 macrophages and provided an advantage over classic inos, arginase1 and cd206 phenotype markers. American journal of respiratory cell and molecular biology. The identity of the mo segregated from subcultured liver, spleen, lung, and brain. These cells are mostly t cells positive for cd3 and cd5 accounting for. Data were collected with flow cytometer lsrii and analyzed with flow jo software. Leukocytes can be obtained from whole blood and a variety of tissues, such as spleen, lymph node, bone marrow and. It is capable of rapid, quantitative, multiparameter analysis of heterogeneous cell populations on a cellbycell basis single cell analysis. Sometimes you just want to know the few, essential markers for phenotyping and identifying your immune cells in flow cytometry rather than every cd marker it expresses. Feb 06, 2019 each macrophage subpopulation in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry.
Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. F480 macrophage marker and the inability to further subdivide the. They acquire different phenotypes and such as langerhans cells in the skin. These cells are strongly adherent to the tissue and thus difficult to analyze by flow cytometry fcm. Characterization of murine macrophages from bone marrow, spleen. Monocytes lack expression of the f480 molecule as well as t, b, and nk cell lineage markers cd3. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. Flow cytometry analysis of bone marrow and spleen cells from wild type wt c57bl6, igmtg and iggtg mice. I wanted to know which marker i should use to sort the splenic macrophages from mice by flow cytometry. To support and simplify the detection of these critical.
Immunophenotyping monocytes, macrophages and granulocytes in. Preparation of human peripheral blood mononuclear cells pbmc leukocytes are the most commonly analyzed cells in flow cytometry. Flow cytometric gating for spleen monocyte and dc subsets. Several tissues including the spleen, lung, central nervous system, liver, skin and bone contain more than one phenotypically distinct macrophage population, which may have different origins. Leukocytes are the most commonly analyzed cells in flow cytometry.