Culture and characterisation of peripheral blood monocytes. Data examples continued this package insert must be read in its entirety before using this product. Improving the methods for isolation of monocyte and establishing. Regulation of monocyte cell fate by blood vessels mediated by. Culture of human monocytederived macrophages springerlink. Cultures can be maintained by the addition of fresh medium or replacement of medium. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with mirna mimics and inhibitors as well as with sirnas. Comparative genotypic and phenotypic analysis of human. Macrophage passaging protocol innovative cell technologies. The in vitro generation of monocyte derived macrophages allows to investigate their role in numerous research applications. Reis e sousa and colleagues show that monocyte derived macrophages resembling immature dcs also develop in such cultures, which should be taken into account when using gmcsf cultures to study dc biology. Muredach reilly lab by hanrui zhang pbmc isolation the 8 ml bd vacutainer mononuclear cell preparation tube cpt with sodium citrate bd, franklin lakes, nj for the separation of mononuclear cells from whole blood was used for blood. Human peripheral blood mononuclear cell pbmc manual.
Mouse monocyte subset phenotypic data are derived from refs 16,22,25,35,94,95. Add 5ml of accutase to the flask or dish and incubate at room temperature for 1015 minutes. Generation of modcs from freshly isolated cells fig. Monocytes are a heterogenous cell population that stem from a precursor gmp common to the neutrophil precursor. Pbmc or monocyte differentiation to macrophage and polarization dr. Dendritic cells can survive in culture for approximately one week. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 24 x 10 5 viable cellsml. Assayready monocyte derived human m1m2 macrophages cryopreserved human.
Do not allow the cell concentration to exceed 1 x 10 6 cells ml. The culture of human monocyte derived macrophages mdms represents a tool to study macrophages, with monocytes known to give rise to tissue macrophages influenced by certain environmental cues. An in vitro monocyte culture method and establishment of a. Apr 01, 2004 in addition, we demonstrate that the differentiation of monocytes into dcs involves an early monocyte activation phase paralleled by the induction of cd11c expression and could be achieved only 24 hours after culture with gmcsf plus il4, whereas longer culture periods appear to be required in the human system. These blood cells are a critical component in the immune system to. While in humans the level of cd14 expression can be used to differentiate nonclassical and intermediate monocytes, the slan cell surface marker was shown to give an unequivocal separation of the two cell types. Tumorderived retinoic acid regulates intratumoral monocyte. Standard protocols for generation of monocytederived cell types. Variables in the isolation and culture of human monocytes that are of particular relevance to studies of hiv. The method involves isolation, culture, and transfection of monocytes and monocyte derived macrophages. To systematically evaluate these immortalised cells and pbmcs as model systems to study inflammation relevant to the pathogenesis of type ii diabetes and immunometabolism, we compared mrna expression of inflammationrelevant. Cfc assay methylcellulose this protocol outlines the general colonyforming cell cfc assay using methylcelulose. The specific methylcellulose formulation can be altered to investigate the effects of drugs andor cytokines on colony formation.
Gently shake and discard nonadherent cells by washing twice with prewarmed culture medium. Pbmc or monocyte differentiation to macrophage and polarization. Ttspecific tcell clones were generated from pbmcs cultured in rpmi5% abs in the presence of 0. Mice that lack mcsf or mcsf receptor have very few circulating monocytes 3, 4. For a more detailed definition of the cell surface receptor and functional heterogeneity of human monocytes, see ref. Culture and differentiation of monocyte derived macrophages using. However, it was assumed these cells belong to the nonmacrophage population due to the nature of the isolation protocol which resulted in a culture containing adherent lymphocytes as well as monocytesdendritic cells. Subculture when cell concentration reaches 8x10 5 cellsml. T lymphocytes isolated from whole blood and cultured in the presence of il2 or il15 for 6 days were allowed to adhere and migrate on glassbottomed dishes coated with 10. Acknowledgements to silvia a cardoso and jorge c andrade for technical assistance. In conclusion, the method described here is simple and reproducible for monocyte isolation and macrophage cell culture of caprine model. Cell culture protocols thermo fisher scientific uk.
Dendritic cell differentiation potential of mouse monocytes. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 24 x 10 5 viable cells ml. Il2 1 uml was added at day 7 of culture, cells were expanded for a further 5 days and then frozen in liquid n 2. Harvest monocytes by short centrifugation and continue as follows. During culture, monocytes increased in size, had increased membrane activity as visualized by sem, and differentiated into a morphologically heterogeneous population of fusiform and epithelioid shapes.
This document describes standard research protocols used for generation of human monocytederived macrophages and dc in the. High throughput, multiplexed detection of inflammatory. Promocell provides a complete macrophage cell culture portfolio including macrophage generation media, cryopreserved human macrophages, cytokines and cell biology tools for subsequent analysis. They will no longer divide, will adhere, metabolise more slowly and take on an appearance that is closer to that of a real macrophage. Remove the supernatant from the flask t25 or tissue culture dish 15mm. High throughput, multiplexed detection of inflammatory cytokines in an astrocyte and monocyte coculture model application note biomarkers, cell imaging, cellbased assays biotek instruments, inc.
In mice and humans, there is good evidence that blood monocytes consist of. A small number of cells were observed to lift after the initial adherence. How to count and calculate the number of cells from a stock flask or culture dish. Cells should be maintained in t75 flasks between 1 x 105 to 4 x 105 viable cellsml and subcultured to maintain this cell density in culture sustaining the cells in a logarithmic phase of growth. A simple protocol for isolating mouse lung endothelial cells. Other data are taken from the references cited i n the main text.
Note that after 5 days of culture in serum or mcsf, your monocytes will be considered macrophages. Pdf culture and differentiation of monocyte derived macrophages. Please make sure that all components you use for dendritic cell culture and transfection e. Typical easysep direct human monocyte isolation profile. Please follow the outlines given in this protocol carefully to ensure reproducibility. Note the multiple dendritelike structures arising from the surface of this nonadherent cell. These ttresponsive cell clones were thawed and expanded for 3. Culture and identification of human monocyte derived. Food and drug administration fda regulations, these products are not for. Cell numbers were insufficient for further characterisation.
For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with mcsf to induce their differentiation into macrophages. Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. Elicitation and collection of peritoneal macrophages and neutrophils using biogel. Our protocol isolates monocytes directly from human peripheral blood by negative. Purified pbmc followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment monocytes increase.
Generation of monocyte derived dendritic cells modcs. Thereafter, monocytes either float or can be detached easily, whereas contaminating cells e. Human t lymphocyte isolation, culture and analysis of. Do not allow the cell concentration to exceed 1 x 10 6 cellsml. Reagents and materials for cell culture included the following. A protocol is presented for cell culture of macrophage colonystimulating factor mcsf differentiated human monocyte derived macrophages. Here we represent an optimized simple method for mdm preparation from peripheral blood mononuclear cells pbmc. A peripheral blood mononuclear cell pbmc is defined as any blood cell with a round nucleus i. An in vitro monocyte culture method and establishment of a human monocytic cell line k63. Aug 31, 2016 ly6c lo monocyte deficiency could be due to increased cell death, as was shown in mice deficient for the transcription factor nr4a1, which controls ly6c lo monocyte numbers in part by regulating.
The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture. The method involves isolation, culture, and transfection of monocytes and monocyte. Add 10 ml of culture medium with a final concentration of 20 ngml and gmcsf 20 ngml il4. Isolation and differentiation of monocytesmacrophages from. This protocol focuses primarily on blood dendritic cells. Monocyte like cell lines mclcs, including thp1, hl60 and u937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells pbmcs. To remove the primary macrophage cultured cells from the tissue culture plates for further analysis. Culture and identification of human monocyte derived dendritic cells subject. Isolation, transfection, and culture of primary human. Human monocyte primary culture im starting a new project and being new on the field of inflammation, id like to know if there are protocols for cultivating human monocyte in vitro and if it is. Culture, freezing and defrosting murine bone marrow macrophages.
In brief, mice lung tissues were harvested, minced, and digested in 3 mgml collagenase i for 45 min, before filtering. The stepbystep procedure of mlecs isolation and culture is shown in fig. Wash the plate or dish with 5ml sterile pbs and remove it. A simple method for human peripheral blood monocyte isolation. A simple and efficient monocyte isolation protocol from human. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. All media, supplements, and tissue culture ware used in this protocol should be sterile.
Culture of bone marrow cells with cytokine gmcsf is a widely used protocol to generate mouse dendritic cells dcs. These cell types retained the ability to phagocytose e glut and ea and to rosette with ea and eac. Just like monocytes, dendritic cells can be difficult to collect following culture depending on the mixture of adherent and nonadherent cells. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. Subculture when cell concentration reaches 8x10 5 cells ml. Box 998, highland park, winooski, vermont 054040998 usa phone. Tips and tricks for growing this popular, but finicky, human monocyte like cell line. A peripheral blood mononuclear cell pbmc is defined as any blood cell with a round nucleus.
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