This protocol describes the procedures for isolating the mononuclear. Functional assays byflow cytometry purdue university. Fresh peripheral blood mononuclear cells were plated in the monocyte attachment medium. Macrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. Abstract originally developed in the late 1960s, flow cytometry is a popular analytical cellbiology technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Until recently, multicolor flow cytometry has been the analytic modality of choice, but it has been limited to measuring approximately 8 parameters because of the need to account for spectral overlap. Analysis of microglia and monocytederived macrophages. Gates are boundaries placed around cell populations that have common features like scatter or marker expression to quantify and study these populations. With a large sensitivity range for fluorescent probes, 95% sampling efficiency, and the ability to sort populations of interest for further study, fluorescentbased cytometry remains a tool of choice for t cell. Characterization of immune cells in human adipose tissue by. Acute monocytic leukemia amol, or amlm5 is considered a type of acute myeloid leukemia. Jci insight cytometry tof identifies alveolar macrophage. Current analysis of macrophage phenotype by flow cytometry is not optimal due to the intracellular location of most m1m2 macrophage markers and the low specificity of available antibodies. We use cd86 gl1, tonbo and cd206 c068c2, biolegend respectively as m1 and m2 markers for flow cytometry.
Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Macrophages, by definition, are a type of immune cells capable of engulfing and digesting various particles in a process called phagocytosis. Flow cytometry analysis for detection of brdu intake was performed after the mice. Need to define the cell surface markers for macrophage phenotype. Arginase1 is upregulated not only in the expected m2 macrophages but also in m1 spectrum macrophages. Flow cytometry fcm is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles in this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrumen. We also offer kits and sets to streamline workflow.
The expression of cd206 and other m2like macrophage markers were also downregualted in iwat of dttreated. Choose from 292 different sets of flow cytometry flashcards on quizlet. Download our latest panels pdf october 2016 standard immunophenotyping panels accurate immunophenotyping by flow cytometry can uncover the mechanism of immunomodulatory treatments. Flow cytometric analysis of inflammatory and resident myeloid. Indepth characterization of monocytederived macrophages. Imbalance of m1m2 macrophages is linked to severity level. Macrophages are very important cells of our immune system. Cellxvivo human m2 macrophage differentiation kit cdk0. This journal also covers relevant techniques such as flow cytometry, image cytometry, beadbased and slidebased array analyses, as well as other. Monocytes and macrophages are phagocytes, acting in both nonspecific defence or innate immunity as well as specific defense or cellmediated immunity of. These cases contain a detailed explanation about each tube and what are the key features to look for on each tube. 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. Phenotypic, functional, and plasticity features of classical. The cluster of differentiation cd is a protocol used for the identification and investigation of cell surface molecules present on leukocytes.
Indepth characterization of monocytederived macrophages using a mass cytometry based phagocytosis assay. 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. Novel markers to delineate murine m1 and m2 macrophages. Cluster of differentiation cd markers flow cytometry. Flow cytometry provides the ability to type immune cells based on their phenotype. In addition, macrophages also exhibit a phenotypic heterogeneity dependent on their local environment. Ijmlr invites articles for vol 5, issue 1, janapr 2020. Flow cytometric assessment of monocyte activation markers and circulating endothelial cells in patients with localized or metastatic breast cancer. Flow cytometry is a laboratory method used to detect, identify, and count specific cells. This information is based on physical characteristics andor markers called antigens on the cell surface.
The following is an introduction to the principles of flow cytometric analysis of hematolymphoid neoplasia. The entire interpretation of flow cytometry data analysis is built upon gating. Clinical cytometry focuses on the development and applications of cellular system analysis and array based technologies as applied to clinical practice and translational research. Characterization of immune cells in human adipose tissue. Flow cytometric analysis furnishes important insights into the immune status of a given tissue by providing information about the numbers and phenotypes of the immune cells that the tissue contains. On day 6 of the differentiation, cells were harvested and stained with antibodies for cd14, cd80, cd163, and cd206 open histograms. Function macrophages may have different names according to where they function in the body.
A protocol for the comprehensive flow cytometric analysis of. We validated this panel using normal lung tissue and tissue from patients with chronic obstructive pulmonary disease and lung fibrosis. Flow cytometry and highcontent imaging to identify markers of monocyte macrophage differentiation. However, differences among macrophages from these particular sources remain unclear. This page illustrates their development and function, as well as the potential role of different subsets. Macrophage infiltration of rat intracranial c6, 9l, and rg2 gliomas was quantified by flow cytometry as described. These cells were first described in 1882 by russian scientist elie metchnikoff, for which he was awarded with the nobel prize in 1908.
Our new page lists the hallmark markers typically used to phenotype several popular immune cells in research. All answers 7 cd16 ie the lowaffinity fc receptor for immunecomplexed igg fc gamma riii is a classic macrophage surface marker that should work, depending of course from which cells you need to differentiate your macrophages from. Learn flow cytometry with free interactive flashcards. Macrophages are found throughout the body in all tissues where they have a critical role in immune surveillance. Amol acute monoblasticmonocytic leukemia m5 flow cytometry. The major difference between other techniques and those proposed using flow cytometry is the ability to. Flow cytometry fcm is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles in this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. In order to differentiate between human monocytes and monocytederived macrophages, which markers can best be used in flow cytometry. Flow cytometric analysis of blood monocytes and alveolar.
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. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. Multicolor flow cytometry resourcestools resources. Flow cytometric analysis of macrophages and dendritic cell. They can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as cd14, cd40, cd11b, cd64, f480 miceemr1 human, lysozyme m, mac1mac3 and cd68. This method can also identify particular components within cells. Cd68 is used primarily in histological staining to identify macrophages, but has been used for this purpose in some flow cytometric studies. The presence of specific cell surface markers, cytokine expression, or phosphorylation of key proteins may be used to immunophenotype specific subpopulations from a heterogeneous starting population. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. 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. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. They acquire different phenotypes and such as langerhans cells in the skin.
For flow cytometry i would have to detach the macrophages from the surface of tissue culture wells and in doing so, i had tried trypsin but was not satisfied with the results. Phenotypic, functional, and plasticity features of. A portion of these cells expressed cd1a 22% and my4 60%. Flow cytometry counting of bronchoalveolar lavage leukocytes. I need some help for finding a good macrophage marker for rats. One of the most common applications is in the diagnosis of leukemia and lymphoma. This tnkupj antibody can be used with the below nonexclusive list of mouse markers table 1. 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 programs leading to an activated state. What is the key marker of macrophage at different phenotype. For each of these cd markers, we strive to provide antibodies tested in several applications to guarantee sensitivity and specificity.
The labs fresh tissue panel is identical to the above, but omits tubes 6, 7, and 8. Standard panels for immunophenotyping flow cytometry. 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. All mi bioresearch standard immunophenotyping flow cytometry panels are copywrited. A group of macrophages, named dendritic cells, acquires the capacity of antigen presentation to the cognate immune system. 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. These cells are strongly adherent to the tissue and thus difficult to analyze by flow cytometry fcm. Surface markers such as cd14, cd206, and cd163 rendered macrophages phagocytosis competent, but only cd209 directly correlated with the amount of particle uptake. Mar 03, 2016 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. Percentages of cells expressing several membrane markers were analyzed. 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. Which marker is best for human macrophages in flow cytometry.
Carter, and padmakumar narayanan cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry. Flow cytometric analysis furnishes important insights into the immune status of a given tissue by providing information about the numbers and. Flow cytometric assessment of monocyte activation markers and. Molecular imaging offers a comprehensive line of antibody panels that enables identification and functional characterization of myeloid and lymphoid subsets that are key regulators of disease.
Flow cytometry and highcontent imaging to identify. Jessica breznik and christian schulz, january 5, 2018 bowdish lab, mcmaster university hamilton, on, canada. Multicolor flow cytometric analysis of sphase brdu incorporating cells using the bd facsarray bioanalyzer. Flow cytometry cpt code 88182 88189 covered dx flow cytometry is a highly complex process by which blood, body fluids, bone marrow and tissue can be examined.
Flow cytometry antibodies bd biosciences offers a wide array of both purified and conjugated antibodies, available in multiple sizes, from sample to bulk. Macrophage markers there are a large number of commonly used macrophage markers such as cd14, cd16, cd64, cd68, cd71 and ccr5. Cd molecules can act in numerous ways, often acting as receptors or ligands the molecule that activates a receptor important to the cell. Jul 01, 20 in a blandlooking leukemia case, for example, if you did flow cytometry and saw that the cells expressed cd and cd 33, youd know the cells were myeloid, and that it was most likely an acute myeloid leukemia. I have an user that needs to sort macrophages and microglia from injured spinal cord in rats. Construction of multicolor antibody panels for the flow cytometric analysis of murine thymic stromal cells. Common macrophage markers tissueresident macrophages are a heterogeneous group of cells that express a variety of cell surface and intracellular markers. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. Flow cytometry is a laboratory method used to detect, identify, and count specific cells from blood, bone marrow, body fluids such as cerebrospinal fluid csf, or tumors. The antibodies and combinations were selected based on the preference of one particular laboratory. It also provides a list of available reagents from biolegend and recommended reading. 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. Our monoclonal antibody tnkupj recognizes mouse cd163 and can be added to any panel aiming to characterize macrophages. The cd in the name of these markers, by the way, stands for cluster designation.
Flow cytometric characterization of tumorassociated. Provides absolute counts, comprehensive analysis for 11 lymphoid and myeloid subsets, and t cell activation analysis. The human cell line thp1 has a monocytic phenotype, including the ability to differentiate into macrophages, providing a tractable, standardised surrogate for human monocytederived macrophages. However, the variation in the case of expression of each marker between patients was large data not shown, thus we calculated the total of m1 and m2 macrophages in each group. It provides important immunophenotypic and dna cycle information, of both diagnostic and prognostic interest in hemopathology, cytopathology and general surgical pathology. It has been successfully used for many decades to accurately visualize and enumerate a variety of t cell subsets. The high autofluorescent cell fraction represented alveolar macrophages which were strongly positive for aph, hladr, cd68, rfd7, and rfd9. Introduction to flow cytometric analysis flow cytometry. Standard panels for flow cytometry established mi bioresearch. Although their markers frequently differ based on their anatomical locations and functions, there is some overlap in the markers that are expressed on different macrophage subpopulations. Common cell surface markers used to identify human and mouse macrophages include cd11bintegrin alpha m, cd14, cd68, fc gamma riiicd16, fc gamma ricd64, and ccr5, along with f480 in mouse. Above is an example panel used for peripheral blood and bone marrow samples. Flow cytometry surface stain for monocytes created by.
If incorporating antimouse cd163, clone tnkupj into a flow cytometry panel, what other markers would be recommended for use and why. There is currently no standard recommended set of marker combinations. The bethesda consensus does provide recommended markers for each category of case types. Chicken cd marker antibodies, cd4, cd8a, cd45 and fluorescent labels suitable for flow cytometry, elisa, western blotting, immunohistochemistry. The product note for the cd206 antibody recommends permeabilizing the cells to be stained but this is unnecessary in my hands for bmdm. Surface phenotyping of human alveolar macrophages by flow cytometry is difficult because of the high autofluorescence of unstained cells. 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. Phagocytosis, endocytosis, and receptor internalization. Flow cytometry is a mainstream tool for studying the immune system. How to use flow cytometry to correctly define t cell. Exemplary flow cytometry analysis of day 10 m1macrophages generated in the promocell m1macrophage generation medium dxf. The removal of dying cells is, to a greater extent, handled by fixed macrophages, which will stay at strategic locations such as the lungs, liver, neural tissue, bone, spleen and connective tissue, ingesting foreign materials such as pathogens and recruiting additional macrophages if needed.
Cells are first gated on the basis of their scatter properties. 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 it is capable of rapid, quantitative, multiparameter analysis of heterogeneous cell populations on a cellbycell basis single cell analysis. Characterization of human peritoneal monocytemacrophage. Cytometry distinguishing m1 and m2 murine macrophages. As compared with microglia, macrophages were less frequent in the tumors and tumor periphery tp and were nearly absent in the contralateral tumorfree hemispheres ch. We have several normal cases posted that you can download and view using the fcs express reader. The table below shows some of the key monocyte markers for humans and mouse. Differentiation of m1 or m2macrophages from pbmcmonocytes. Furthermore, the intensity of autofluorescence in lung macrophages can interfere with spectral analysis. It is important to note that these pancancer antigens are not true cell lineage markers and though a tumor may stain positively by immunohistochemistry or flow cytometry for an antigen, every cell in that lesion may not express the target antigen, leaving a population inherently resistant to car t cell therapy. 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. 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. When a macrophage ingests a pathogen, the pathogen.
Analysis of single and doublestained alveolar macrophages by. There are very few unique macrophage markers and often a number of markers will be required to identify your cell type. Which markers are best to differentiate between human. Additional markers are used to identify specific tissueresident macrophages and different macrophage activation states. Flow cytometry is used extensively to examine immune cell repertoires and follow immune responses in nonlymphoid tissues. Aug 24, 2018 flow cytometry analysis of phenotypic characteristics of human peritoneal monocyte macrophages compared to blood monocytes. Flow cytometry is a remarkably powerful tool for the study of t cells. Nov 11, 2019 these markers can be identified using a technical process called flow cytometry. It is capable of rapid, quantitative, multiparameter analysis of heterogeneous cell populations on a cellbycell basis single cell analysis.
Flow cytometry based analysis and sorting of lung dendritic cells 241 cd68 van haarst et al. Flow cytometry analyses for expression of cd11c as m1 and cd206 as m2 marker exhibited that both m1 and m2 macrophages were present in the synovial fluids from knee oa. A protocol for the comprehensive flow cytometric analysis. Macrophages are key in orchestrating immune responses to microenvironmental stimuli, sensed by a complex set of surface receptors. Cell viability for flow cytometry cytotoxicity ion indicators oxidative stress detection phagocytosis, endocytosis, and receptor internalization phagocytosis endocytosis and pinocytosis. Within a population of ham from a single donor there is wide variation in autofluorescence and the range of autofluorescence also varies between donors. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to. 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. Characterization of murine macrophages from bone marrow. In order to fulfill world health organization who criteria for amlm5, a patient must have greater than 20% blasts in the bone marrow, and of these, greater than 80% must be of the monocytic lineage.