title: Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.
abstract: Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Menck
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen.
authors.forename: Daniel
authors.initials: D
authors.lastname: Behme
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen.
authors.forename: Mathias
authors.initials: M
authors.lastname: Pantke
authors.affiliation: Microbial Interface Biology Group, Research Center Borstel.
authors.forename: Norbert
authors.initials: N
authors.lastname: Reiling
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen.
authors.forename: Claudia
authors.initials: C
authors.lastname: Binder
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen.
authors.forename: Tobias
authors.initials: T
authors.lastname: Pukrop
authors.affiliation: Department of Hematology and Oncology, University Medical Center Göttingen; florian.klemm@med.uni-goettingen.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Klemm
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002499:Centrifugation, Density Gradient / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D008264:Macrophages / Q000166:cytology*; D009000:Monocytes / Q000166:cytology*; D011138:Polytetrafluoroethylene / Q000737:chemistry
chemical_list: D011138:Polytetrafluoroethylene