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. Screen pmadifferentiated thp1 cells adherent macrophages stained with the bd. Our monoclonal antibody tnkupj recognizes mouse cd163 and can be added to any panel aiming to characterize macrophages. Flow cytometry has been used to reliably quantify phagocytosis of bacteria, malarial parasites, liposomes, zymosan particles, latex beads, unopsonised environmental particles, platelets, and erythrocytes 19.
Distinct subpopulations of macrophages can be defined by their tissue of residence and functional phenotype. Flow cytometry protocols for cell membrane marker protein staining. We also recommend flow cytometry controls, instrument setup, and the determination of positivity. 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. The following flow cytometry staining protocol has been. Staining intracellular antigens for flow cytometry research use only.
Select mouse tissueresident macrophage populations that have been identified include. Can anybody advise me which stimulation protocol is more suitable and. Jessica breznik and christian schulz, january 5, 2018 bowdish lab, mcmaster university. Jurkat cells were either unstimulated or treated with pma 50 nm, or pma 50 nm and. Macrophages facilitate electrical conduction in the. Macrophages, by definition, are a type of immune cells capable of. The macrophages were resuspended in pbs containing 2% fetal bovine serum fbs. Flow cytometry and highcontent imaging to identify markers of. Characterization of mouse adult testicular macrophage bioprotocol. A detailed description of flow cytometry immunophenotyping, including links to information on. Screen pma differentiated thp1 cells adherent macrophages stained with the bd. Bcell analysis, seven cell surface markers were used to analyze bcell subsets in mouse bone marrow, allowing discrimination of seven different developmental phases in this tissue.
Stop cell lysis by adding 10ml cell staining buffer to the tube. Phenotypic characterization of macrophages from rat kidney. Would the above markers be enough to differentiate between m1 and m2 in flow cytometry. Characterization of murine macrophages from bone marrow. Centrifuge for 5 minutes at 350xg and discard supernatant. As this protocol is based mainly on flow cytometry, it is crucial to verify the proper function and setup of the flow cytometer or cell sorter by running an appropriate cytometer procedure. Phenotyping cells by flow cytometry is a powerful way to identify cell type and any morphological changes during cell culture. A protocol for the comprehensive flow cytometric analysis. Cell surface markers are expressed on the cell surface and can be used to. The interactive cell markers page shows various cell types and the cell surface markers associated with that cell. Phagocytosis of apoptotic cells by human macrophages. 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. Characterisation of lung macrophage subpopulations in copd.
Here we report a flow cytometry protocol that allows unambiguous identification of alveolar macrophages, interstitial macrophages, and monocytes in the human lung and in. 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. American journal of respiratory cell and molecular biology. Protocol cell surface flow cytometry staining protocol. Monocytes, macrophages, granulocytes, platelets, myeloid dendritic cells. Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Thp1 cell surface markers for flow cytometry need a robust marker for undifferentiated thp1 cells sep242009 i am working with thp1 cells atcc tib 202, a human monocytic cell line. Reports of flow cytometry data for publication should include information on the antibodies clone, commercial.
Count viable cells and resuspend in cell staining buffer at 510 x 10 6 cells ml and distribute 100ltube of cell suspension 510 x 10 5 cells tube into 12 x 75mm plastic tubes. Flow cytometry is used extensively to examine immune cells in nonlymphoid tissues. Flow cytometry is used extensively to examine immune cells in. 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. The quantification of cell surface antigens on human alveolar macrophages using flow cytometry is complicated by strong autofluorescence which varies with cell size and granularity. Fluorescent spectraviewer supports flow cytometry, fluorescence microscopy. Identification and isolation of splenic tissueresident. This is one of the simplest and most common staining methods. Displays useful information such as other names, structure, distribution, function, and ligand. By staining cell surface markers, researchers can identify specific cell populations and perform fluorescenceactivated cell sorting facs. Analysis of single and doublestained alveolar macrophages. Cell surface markers are expressed on the cell surface and can be used to define cell subtypes as well as function when they are labeled with fluorescentlabeled antibodies and analyzed by flow cytometry. What is the best procedure for fixation of cells when antibodies for. Since i am extracting my cells from organ, i need to facs it.
We developed a flow cytometry protocol to identify and sort microglia. Flow cytometry analysis reported low cell mortality during the isolation protocol. Stain cells with surface markers, wash, fix, permeabilise and then. Flow cytometric analysis of macrophages and dendritic cell. Flow cytometry protocol flow cell signaling technology. To support this effort, cell surface markers are provided for standard pulmonary cell populations. 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 recent perspectives 240 sterile surgical blades vwr. View are there any specific cell surface markers for m1 and m2 macrophages. Standard protocols for flow cytometry for monocytes, macrophages. Treatment of monocytederived macrophage and flow cytometry analysis of cell surface expression markers human monocytederived macrophages, obtained as already described, were resuspended at.
Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. General procedure for flow cytometry using a conjugated primary antibody print this protocol. A protocol for the comprehensive flow cytometric analysis of. Whats the best marker in flow cytometry to differentiate. We prepared single cell suspensions of lung cells in flow cytometry staining buffer consisting of 10% fbs supplemented pbs. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in flow cytometry. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. The cluster of differentiation also known as cluster of designation or classification determinant and often abbreviated as cd is a protocol used for the identification and investigation of cell surface.
Analysis of microglia and monocytederived macrophages. This method is suitable for cell surface staining in flow cytometry. If incorporating antimouse cd163, clone tnkupj into a flow cytometry panel, what other markers would be recommended for use and why. Intracellular staining flow cytometry protocols in order to detect antigen not present on the cell surface, cells have to be fixed and permeabilized to disrupt the cell membrane and allow entry of the antibody. The mycotoxin deoxynivalenol inhibits the cell surface. Misharin av1, moralesnebreda l, mutlu gm, budinger gr, perlman h. Flow cytometry is a technique for counting particles using electronic detection apparatus, and is often used to collect quantitative information about cell populations. Antigens can be then directly or indirectly labeled. Cell surface markers can be used to define cell subsets based on lineage and developmental stage, as well as function when they are labeled with fluorochromeconjugated antibodies and analyzed by flow. There are several factors that may affect cell viability and surface markers detection, such as the level of tissue. Harvest, wash the cells and adjust cell suspension to a concentration of 15 x 10 6 cellsml in ice cold pbs. Macrophage, testis, facs, if, spermatogenesis, immuneprivilege organ. Flow cytometry is used extensively to examine immune cell repertoires and follow immune responses in nonlymphoid tissues. Various methods are optimal depending on the antigen and antibody used.
The phenotype of mature tissue macrophages represents the composite of environmental. Flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung. 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. Nonspecific ab binding was blocked with addition of fc block ab, then. This would help to discriminate your macrophages further in the spectrum of m1 to. Whats the best marker in flow cytometry to differentiate between m1.
Thp1 cell surface markers for flow cytometry flow cytometry. Exemplary flow cytometry analysis of day 10 m1macrophages generated in the promocell m1mac. Which marker is best for human macrophages in flow cytometry. We then analyzed the cell surface antigen expression on each enzymedissociated macrophage subpopulation by means of flow cytometry. Flow cytometry surface stain for monocytes created by. The staining procedure used in this chapter enables the. What is the best protocol to obtain bone marrow derived macrophages bmdm. The entire interpretation of flow cytometry data analysis is built upon gating. Surface marker analysis by flow cytometry thermo fisher. This tnkupj antibody can be used with the below nonexclusive list of mouse markers table 1.