This cellular lineage of macrophages, the mononuclear phagocyte. Flow cytometry antimacrophage antibody bm8 phycoerythrin ab125148 c57bl6 bone marrow cells were stained with apc antimouse cd11b ab25482 and 0. The markers used were cd45 apc fluorophore, immunotools, cd14 pe fluorophore, immunotools. There are a large number of commonly used macrophage markers such as cd14, cd16, cd64, cd68, cd71 and ccr5. B scatterplots from mcsfstimulated mdms incubated with oso 4. Mesenchymal stem cells induce suppressive macrophages. E the impact of cd16 blockade on phagocytosis was assessed by flow cytometry. If incorporating antimouse cd163, clone tnkupj into a flow cytometry panel, what other markers would be recommended for use and why. For instance, there are no human homologs of the mouse m2 markers ym1. The percentage of rbcs phagocytosed by monocytes was determined on the basis of double positivity for both markers. A group of macrophages, named dendritic cells, acquires the capacity of antigen presentation to the cognate immune system. 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.
Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Flow cytometry is a mainstream tool for studying the immune system. For the phagocytosis assay simply use the standard protocols for flow cytometry which is use fitc grown cultures of e coli and incubate a relation of to 0 bactera per macrophage in a. Is there a phagocytosis assay using flow cytometry. Macrophages are a heterogeneous population of innate myeloid cells involved in health and disease. A schematic of the mass cytometrybased phagocytosis assay. 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. Phenotypic characterization of macrophages from rat kidney. Phagocytosis of bacteria has been well characterized by bassoe and bjerknes using flow cytometry 4, 5. A flow cytometry protocol is available that provides detailed procedures for the treatment and. A source of phagocytic cells such as human pbmcs, mouse bone marrowderived macrophages, or cell lines like raw 264.
Many tissueresident macrophages with embryonic origins can selfrenew and persist into adulthood, while those in other tissues are gradually replaced by bone marrowderived monocytes. Which marker is best for human macrophages in flow cytometry. The markers used were cd45 apc fluorophore, immunotools, cd14 pe fluorophore, immunotools, cd11b apc fluorophore, immunotools. Indepth characterization of monocytederived macrophages. Frontiers cd5l promotes m2 macrophage polarization. Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. In a new, simple, and fast flowcytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the wellknown vital stain, neutral red was used. Polarization of rheumatoid macrophages by tnf targeting. There are very few unique macrophage markers and often a number of markers will be required to identify your cell type. Tissueresident macrophages have central roles in tissue development and homeostasis, immune surveillance, phagocytosis, antigen presentation, and wound healing.
The data shown are from 514 independent experiments and indicated that phagocytosis is not maximal at the 30. A schematic of the mass cytometry based phagocytosis assay. The main cell surface receptors mediating phagocytosis are c3b crl and fcr fc portion of immunoglobulin g. Flow cytometry based on cd38egr2 can distinguish m1 and m2.
The markers that best characterized m1 macrophages were cd64 the highaffinity fc. 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. The phrodo bioparticles phagocytosis assays provide sensitive detection without the. Hoang nguyen research fellow mskcc next part html attachment scrubbed and removed.
A protocol for the comprehensive flow cytometric analysis. Fresh peritoneal macrophages were processed for phagocytosis ac or cell surface markers analysis dh. For reproducibility, the percent positive measure of. Axol ipscderived macrophages were harvested at day 7 using cold 1x pbs containing 5 mm edta. Impairment in phagocytosis has been associated with numerous diseases and disorders. Frontiers cd5l promotes m2 macrophage polarization through. Cd68 antibodies are a vital tool in macrophage research. Detect the expression of macrophage specific markers and polarization markers using gene expression analyses and flow cytometry as previously described 8, 9. Flow cytometry has been used to reliably quantify phagocytosis of bacteria, malarial parasites, liposomes, zymosan particles, latex beads, unopsonised environmental particles, platelets, and erythrocytes 1926.
Phagocytosis was determined by flow cytometry after different periods of coincubation of macrophages with apoptotic neutrophils. Our monoclonal antibody tnkupj recognizes mouse cd163 and can be added to any panel aiming to characterize macrophages. Flow cytometry and highcontent imaging to identify markers. Defective macrophage phagocytosis of bacteria in copd.
In the gated events, phagocytosis percentage and mfi were assessed and uninfected controls were used to determine cutoffs for phagocytosis. Analysis of microglia and monocytederived macrophages from. Macrophage phagocytosis assay of staphylococcus aureus by. Flow cytometry fcm offers rapid and reproducible measurements of single cells in suspension and, following staining with one or more fluorochromes, simultaneous biochemical and functional examinations of the complex process of phagocytosis. Functional assays byflow cytometry purdue university. In order to differentiate between human monocytes and monocytederived macrophages, which markers can best be used in flow cytometry. We then analyzed the cell surface antigen expression on each enzymedissociated macrophage subpopulation by means of flow cytometry. In mice, m1 macrophages can be characterized by expression of inos and m2 macrophages by arginase1 production. We used a flow cytometric method that allows quantification of.
Phagocytosis of phrodo green escherichia coli bioparticles life technologies by alveolar and interstitial macrophages for 1 hour was quantified by flow cytometry as described in the online. Are there any markers of macrophage phagocytic activity. The graphs show the integrated median fluorescence. Consistent with previous reports, we find both mertk and cd169 to be highly specific macrophage markers. This tnkupj antibody can be used with the below nonexclusive list of mouse markers table 1. Macrophages were originally identified by ilya metchnikoff nathan cf 2008 more than 100 years ago, and his description of phagocytosis won him the nobel prize for medicine in 1908. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. However, the importance of single and combinatorial surface marker. Identification and isolation of splenic tissueresident. Receptor expression was measured by flow cytometry. In a facscanto machine, the fluorescence produced from hexidium iodide staining falls into fl2 channel excitation 488emmision 575 and fluorescence produced from fitc staining falls into fl1 channel excitation 488emmision 519. Higher phagocytic activity of thioglycollateelicited.
Preliminary assessment of the flow cytometryphagocytosis assay preliminary results using three human monoclonal antibodies are shown in figures 1 and 2. Novel markers to delineate murine m1 and m2 macrophages. Among the top distinct genes in m1 or m2 macrophages, we validated cd38, gpr18 and fpr2 as novel m1 markers and egr2 and cmyc as m2 markers. Preliminary assessment of the flow cytometry phagocytosis assay preliminary results using three human monoclonal antibodies are shown in figures 1 and 2. Autophagy regulates phagocytosis by modulating the. 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. Evaluation of erythrocyte autoantibodies with flow. Phagocytosis of phrodo green escherichia coli bioparticles life technologies by alveolar and interstitial macrophages for 1 hour was quantified. Indepth characterization of monocytederived macrophages using.
For flow cytometry experiments on one well of a 6 well plate of macrophages, harvest cells by aspirating their maturation media, wash with 2 ml of pbs, and incubate them with 2 ml of. Assessment of phagocytic activity of cultured macrophages. Mdm were activated as m1 macrophages for 24 h in the presence or not of ada and an anticd16 blocking antibody 10. Different cytokines have been shown to affect the phagocytic process. Phagocytosis of apoptotic cells by human macrophages. Flow cytometry based on cd38egr2 can distinguish m1 and m2 macrophages, and has advantages over classical inos, arginase1 and cd206 phenotype markers 26. 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. American journal of respiratory cell and molecular biology. The assay can be used for the rapid and accurate detection and quantification of in vitro phagocytosis by fluorescent microscope, spectrophotometer or flow cytometry. This protocol describes the procedures for isolating the mononuclear. We utilized a method named flow cytometry that is used to measure fluorescence in single cells. 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. Flow cytometrybased phagocytosis assay for sensitive.
May 19, 2014 impairment in phagocytosis has been associated with numerous diseases and disorders. Phagocytosis assay kit ab234054 uses zymosan particles which have been prelabeled with a red dye. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. Jan 18, 2019 d the effect of bdmard on phagocytosis by m1 macrophages was assessed by flow cytometry 5 healthy controls. Surface markers such as cd14, cd206, and cd163 rendered macrophages phagocytosis competent, but only cd209 directly correlated with the amount of particle uptake. Flow cytometry with two distinct fluorescent labels was used for quantification.
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. 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. Since phagocytosis is such a critical function we developed a unique way to test how well rpe cells can phagocytose the tips of the photoreceptor cells before implantation. In a new, simple, and fast flow cytometric method for the simultaneous measurement of phagocytic activity of human neutrophils and monocytes in whole blood, the fluorescence capability of the wellknown vital stain, neutral red was used. Analysis of microglia and monocytederived macrophages. Characterisation of lung macrophage subpopulations in copd.
These cells are strongly adherent to the tissue and thus difficult to analyze by flow cytometry fcm. Validation of surface markers of macrophage polarization. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. I am looking to assess phagocytosis in human macrophages and looking for a reliable phagocytosis assay by flow cytometry. The phrodo bioparticles phagocytosis assays provide sensitive detection without the need for quenching reagents and extra wash steps. Macrophages constitute a very versatile population of phagocytic cells present in most tissues. It provides a robust screening system for activators andor inhibitors of phagocytosis and tolllike receptors ligands tlr. View which are the specific markers of m1 and m2 macrophages by immunohistochemistry. Common cell surface markers used to identify human and mouse macrophages include cd11b integrin alpha m, cd14, cd68, fc gamma riiicd16, fc gamma ricd64, and ccr5, along with f480 in mouse.
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. The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. Evaluation of erythrocyte autoantibodies with flow cytometric phagocytosis assay shoichi ito 1, tomoko hishinuma 1, yoshiko ogiyama 1, tomomi asano 1, haruka kagaya 1, michiyo irino 1, hideya hasegawa 1, hiroshi shimizu 1, kenneth e. Flow cytometry and highcontent imaging to identify. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. We have demonstrated that quantification of macrophage phagocytosis by flow cytometry is a robust and reproducible. This means that robust quantification of rare events during infection is.
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. Flow cytometry for key cell surface markers of innate immunity. Alveolar macrophages and mdm phagocytosed polystyrene beads similarly. Macrophages, by definition, are a type of immune cells capable of engulfing and digesting various particles in a process called phagocytosis.
Therefore, human macrophages from both categories can be characterized by the profile of cytokines produced and the expression of cell surface markers. The possibility of using surface markers to discriminate these different polarization states was assessed by flow. The kit includes all of the reagents required for assessing particle ingestion and red blood cell lysis. A protocol for the comprehensive flow cytometric analysis of. They acquire different phenotypes and such as langerhans cells in the skin. In the most distinct genes in m1 or m2 macrophages, cd38, gpr18 and fpr2 were new m1 markers, and egr2 and cmyc were m2 markers. The present report describes a phagocytosis assay that permits rapid and sensitive quantification of pneumococcal phagocytosis. Cd68 is used primarily in histological staining to identify macrophages, but has been used for this purpose in some flow cytometric studies. Here we developed a novel mass cytometrybased phagocytosis assay. These markers help distinguish macrophages from other similar cell populations. A multicolor flow cytometry analysis of hladr, cd80, cd23, cd206, and cd163 marker profiles in pb monocytes treated for 72 h with medium alone. Pbmc from 16 ra patients and 20 controls were differentiated into mdm and activated or not as m1 proinflammatory macrophages, mil4 alternative macrophages, and mil10 alternative macrophages figure 1a.
Phagocytosis of fluorescently labelled polystyrene beads and haemophillus influenzae and streptococcus pneumoniae by alveolar macrophages and monocytederived macrophages mdm was assessed by fluorimetry and 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 green fitc label reflects the association of bacteria with pmnl, whereas the red label pe detects s. Are there any phagocytosisassociated surface markers of. These cells were first described in 1882 by russian scientist elie metchnikoff, for which he was awarded with the nobel prize in 1908.
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