During development and throughout life, macrophages reside in many tissues of the body, contributing to both the maintenance. The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colonystimulating factor, csf1r. Flow cytometry is a mainstream tool for studying the immune system. Irf5 is a specific marker of inflammatory macrophages in vivo. Our protocol resulted in low cellular mortality and allowed. Subpopulation i included the population that expressed. Early fate defines microglia and nonparenchymal brain. The results of this study and the new markers we have identified, including a new cd38egr2based flow cytometry strategy to distinguish m1 and m2 macrophages, can be found in jablonski et al.
Transcripts of selected flow cytometry webinars thermo. We developed a protocol for the preparation and flow cytometric. In mice, m1 macrophages can be characterized by expression of inos and m2 macrophages by arginase1 production. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. We validated this panel using normal lung tissue and tissue from patients with chronic obstructive pulmonary disease and lung fibrosis. Representative flow cytometry plots of blood monocytes isolated from day 6 dss treated or drinking water controls stimulated with 1. A protocol for the comprehensive flow cytometric analysis. Macrophages are very important cells of our immune system. Macrophages also act as antigenpresenting cells recruited via the inflammatory response, and they act also as independent immune cells in injured organs when a swift and effective response is needed.
We developed a protocol for the flow cytometric analysis of. Blood lymphocyte immunophenotyping by flow cytometry is a routine diagnostic procedure for assessment of lymphoproliferative diseases and hiv patient immunodeficiency. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Therefore, human macrophages from both categories can be characterized by the profile of cytokines produced and the expression of cell surface markers. We use cd86 gl1, tonbo and cd206 c068c2, biolegend respectively as m1 and m2 markers for flow cytometry. Although there is a lot of overlap between these cells types, regarding both cell surface markers and function, there are still some distinct features that can uniquely identify them. Characterisation of lung macrophage subpopulations in copd. A protocol for the comprehensive flow cytometric analysis of. Macrophage subpopulation cellsurface expression of immunological markers and phagocytic ability were assessed by flow cytometry.
This notoriously heterogeneous system is composed of macrophages themselves in addition to dendritic cells, monocytes and their lineage committed progenitors. 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. Flow cytometry and highcontent imaging to identify markers of monocyte macrophage 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. The role of different components of innate immunity, such as monocytes, macrophages, dendritic cells, nk cells, and different innate lymphocytes, and their ability in controlling the early phases of the infection. Both functions are accomplished by a pivotal network of different macrophage. Dengue virus infects macrophages and dendritic cells in a. The product note for the cd206 antibody recommends permeabilizing the cells to be.
Dockrell department of infection and immunity, medical school, university of sheffield, sheffield, united kingdom abstract. These markers help distinguish macrophages from other similar cell. Phenotypic characterization of macrophages from rat kidney. Flow cytometry with adherent cells purdue university. The cells were fluorescently stained with cd45vioblue, cd11bapc, and antif480fitc and analyzed by flow cytometry using the macsquant analyzer. Whats the best marker in flow cytometry to differentiate. Manually selected mucus plug material was treated with dithiothrietol, filtered and total leukocytes acquired. Macrophages are vital for homeostasis via phagocytosis of dead and dying cells. The production and utilization of cytokines, chemokines, and their receptors, which are crucial in the initial cytokine storm. Defining human dendritic cell progenitors by multiparametric. Representative scheme of the different macrophage phenotypes according to different distribution for cd45, cd68, cd86 and cd163 markers c. 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 reveals similarities between lung macrophages.
Confocal images were obtained from lung tissue using antibodies for cluster of differentiation cd206, cd169, and e cadherin. Nov 11, 2019 these markers can be identified using a technical process called flow cytometry. The exact markers expressed by the monocyte will depend upon the subtype of monocyte and the external stimuli, however monocytes often lack lineage markers for t cells, b cells, nk cells and dc cells, such as. We then analyzed the cell surface antigen expression on each enzymedissociated macrophage subpopulation by means of flow cytometry. Flow cytometry and highcontent imaging to identify markers of monocytemacrophage differentiation dev mittar, rosanto paramban, and catherine mcintyre bd biosciences contents 1 abstract 2. Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro and anti. Lymphocyte and monocyte flow cytometry immunophenotyping. Flow cytometry and highcontent imaging to identify markers. Multicolor flow cytometry was performed using specific gating strategies based on light scatter properties, differential expression of cd45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris. The exact markers expressed by the monocyte will depend upon the subtype of monocyte and the external stimuli, however monocytes often lack lineage markers for t cells, b cells, nk cells and dc. Schematic representation of renal cell isolation a and macrophage detection by flow cytometry b. Similarly, cd11b has often been used as a marker for all myeloid cell types.
If we look specifically at this population and we use a marker for m2 macrophages, or tams, tumorassociated macrophages, here in this example, were using cd163 and via flow cytometry, what we see. Flow cytometry and highcontent imaging to identify markers of. Rat markers cd44 and cd4 have been used in flow cytometry figure 1, showing t cell, b cell and macrophage populations. Macrophages also act as antigenpresenting cells recruited via the inflammatory response, and they act also as independent. 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. Functional assays byflow cytometry purdue university. Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of cajal icc and myenteric neurons. More recently it has become part also of the monitoring of patients taking immunemodifying drugs such as the rituximab mabthera anticd20 monoclonal antibody. Historically, m1m2 marker discovery started in mouse macrophages. Which markers are best to differentiate between human monocytes. Cytometry distinguishing m1 and m2 murine macrophages. Facs plots are representative from 1 out of 34 independent experiments. Alveolar macrophages are specialised resident phagocytes in the alveolus, constituting the first line of immune cellular defence in the lung. Jul 19, 2019 the proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colonystimulating factor, csf1r.
This protocol describes flow cytometry panels that can be used to analyze and isolate dendritic cell dc progenitors including granulocyte, monocyte and dc progenitor cells. Flow cytometry and highcontent imaging to identify. Macrophages can be classified into two major subtypes. Flow cytometry analysis of rat makers cd44 and cd4. The objective of this study was to develop a flow cytometrybased panel for human bal and lung tissue. Oct 18, 2016 rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. If we look specifically at this population and we use a marker for m2 macrophages, or tams, tumorassociated macrophages, here in this example, were using cd163 and via flow cytometry, what we see is that we see a 44. Development of monocytes, macrophages and dendritic cells. Change in populations of macrophages promotes development of. Microarray experiments included 4 biologically independent samples each of which containing 4 to 6 e14. Molecular marker development development of molecular markers. Sep 24, 2018 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.
We further confirmed these data by flow cytometry and show that m1 and m2. Which marker is best for human macrophages in flow cytometry. In general, macrophages develop from monocytes in the bone marrow, but they can. Bd solutions, including flow cytometers, software, conjugated antibodies, and services, are designed to support the rapid pace of discovery in tcell research. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells dcs accurately and. Aug 20, 2015 this protocol describes flow cytometry panels that can be used to analyze and isolate dendritic cell dc progenitors including granulocyte, monocyte and dc progenitor cells.
Carter, and padmakumar narayanan cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry. Multicolor flow cytometry was performed using specific gating strategies based on light. Protocol for renal cells isolation and macrophage detection by flow cytometry. Frontiers dynamics of colon monocyte and macrophage. Jul, 2010 blood lymphocyte immunophenotyping by flow cytometry is a routine diagnostic procedure for assessment of lymphoproliferative diseases and hiv patient immunodeficiency.
The kit combines the benefits of flow cytometry and high quality pretitrated antibodies to. Generation, culture and flowcytometric characterization of. Following this, cd4 has been used in immunohistochemistry showing t cells. We have recently developed novel antibodies to both cd163 and mertk to allow new insights into. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. The optimized panel allows for simultaneous detection of 21 myeloid. Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Cd68 is used primarily in histological staining to identify macrophages, but has been used for this purpose in some flow cytometric studies. Lymphocyte and monocyte flow cytometry immunophenotyping as a. The major difference between other techniques and those proposed using flow cytometry is the ability to.
Facs plots are representative from 1 out of 34 independent. Tissueresident macrophages have central roles in tissue development and homeostasis, immune surveillance, phagocytosis, antigen presentation, and wound healing. Finally on many occasions i have wanted to see how flow cytometry results compare with what the cells actually look like so i often culture the cells on microscope slides or on chamber slides, stain and look. Our solutions for tcell identification, transcription factor expression, as well as cytokine secretion and measurement, reflect a commitment to high quality and consistency needed for. American journal of respiratory cell and molecular biology. Identification and isolation of splenic tissueresident. In vitro, monocytes can be differentiated into m1 and m2 macrophages using gmcsf or mcsf, respectively. Nov 27, 2018 representative flow cytometry plots of blood monocytes isolated from day 6 dss treated or drinking water controls stimulated with 1. The product note for the cd206 antibody recommends permeabilizing the cells to be stained but this is unnecessary in my hands for bmdm. Novel markers to delineate murine m1 and m2 macrophages plos. Expression analyses of conventional mo markers by flow cytometry showed similar expression patterns. 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. The identification of markers of macrophage differentiation. Primitive embryonic macrophages are required for coronary.
Phenotypic characterization of macrophages from rat kidney by. This meeting highlighted the large potential of flow cytometric tools available for. Flow cytometry characterization of circulating cells belonging to the monocytemacrophage lineage was performed using specific m1 cd80, cd86, tlr2 and tlr4 and. A new therapeutic pathway in fibrosing lung disease. Function macrophages may have different names according to where they function in the body. Design we studied the expression of homing markers on monocyte subsets in the peripheral blood and on macrophage subsets in the gut of patients with ibd and controls with flow cytometry and. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and dcs, and their. The role of different components of innate immunity, such as monocytes, macrophages, dendritic cells, nk cells, and different innate lymphocytes, and their ability in controlling. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. The table below shows some of the key monocyte markers for humans and mouse. Change in populations of macrophages promotes development.
Flow cytometric analysis of macrophages and dendritic cell. 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. Flow cytometric analysis of inflammatory and resident myeloid. 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. 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. The identification of markers of macrophage differentiation in pmastimulated thp1 cells and monocytederived macrophages marc daigneault, julie a. Using flow cytometry to analyze cryptococcus infection of. Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages, and monocytes in the human lung and in. Many tissueresident macrophages with embryonic origins can selfrenew and persist into adulthood, while those in other tissues are gradually replaced by bone marrowderived monocytes. There are a large number of commonly used macrophage markers such as cd14, cd16, cd64, cd68, cd71 and ccr5.