Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Abstract

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

CD11b+Gr‐1dim Tolerogenic Dendritic Cell–Like Cells Are Expanded in Interstitial Lung Disease in SKG Mice

“…In vitro differentiation of CD11b+Gr‐1 dim cells from monocytic MDSCs by stimulation with GM‐CSF and IL‐4. The generation of DCs from BM cells in vitro using GM‐CSF is well established . To determine whether DCs could be generated from lung cells using GM‐CSF, we cultured total lung cells with GM‐CSF in vitro in the presence or absence of IL‐4.…”

Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

“…The samples were centrifuged, and the supernatant was discarded. The cells were resuspended and cultured in RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL M-CSF (CB34, Novoprotein) [ 20 ]. BMDMs were incubated with APC anti-mouse/human CD11b antibody (101,212, Biolegend) and FITC anti-mouse F4/80 antibody (123,108, Biolegend), identified by flow cytometry.…”

The aryl hydrocarbon receptor regulates lipid mediator production in alveolar macrophages

“…BMDMs of wildtype (WT), AhR KO/KO , Vav iCre x AhR flox/flox and LysM Cre x AhR flox/flox mice were differentiated as described with minor modifications to better mimic alveolar-like macrophages ( 23 , 24 ). Briefly, bone marrow was flushed from femur and tibia, filtered with cell strainers (70 µm) and 1.5 x 10 7 cells were cultured in a TC cell culture flask (T-75, Sarstedt, Nümbrecht, Germany) in 15 ml RPMI-1640 medium (ThermoFisher Scientific) containing 10 ng/mL GM-CSF (Peprotech) and 2 ng/mL TGF-ß1 (Peprotech) to drive differentiation of alveolar-like BMDMs.…”