Murine Monocyte and Macrophage Culture

Abstract

Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past to differentiate myeloid cell types from bone marrow cells, enabling functional investigation and furthering our understanding about their contribution to mammalian physiology. In this protocol, we describe a simple and rapid method to isolate monocytes from murine bone marrow, culture them for up to 5 days and lastly, differentiate them into bone marrow derived macrophages (Figure 1).

SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of Porphyromonas gingivalis

“…Both MC3T3-E1 and GECs were cultured in standard culture medium (DMEM medium supplemented with 10 % FBS and 1 % antibiotics (streptomycin 100 U/mL, penicillin 100 U/mL)). Mice bone marrow-derived macrophages (BMDM) were harvested and cultured [27] .…”

Recruitment of large peritoneal macrophages to capsular fibrosis developed on the liver surface

“…Kupffer cells were isolated from the normal liver by in situ liver digestion followed by Percoll separation 22 . To isolate BM‐derived monocytes, we collected the femur and tibia, obtained cells from the BM by centrifugation, and purified monocytes by MACS using Monocyte Isolation kit (Miltenyi Biotec) 23,24 . To induce BM‐derived monocytes to macrophages in vitro, monocytes were cultured in DMEM containing 10% FBS and 5 ng/mL of mouse recombinant M‐CSF (ThermoFisher Scientific) for 6 days.…”

“…22 To isolate BM-derived monocytes, we collected the femur and tibia, obtained cells from the BM by centrifugation, and purified monocytes by MACS using Monocyte Isolation kit (Miltenyi Biotec). 23,24 To induce BM-derived monocytes to macrophages in vitro, monocytes were cultured in DMEM containing 10% FBS and 5 ng/mL of mouse recombinant M-CSF (ThermoFisher Scientific) for 6 days. Cultured macrophages were collected by digestion with a trypsin-EDTA solution.…”

Erythropoietin Receptor (EPOR) Signaling in the Osteoclast Lineage Contributes to EPO-Induced Bone Loss in Mice

“…Murine monocytes were harvested from femurs and tibias of 12-week-old male and female transgenic mice (Cx3cr1 Cre EPOR f/f mice and their corresponding controls Cx3cr1 Cre ) by negative selection using a monocyte isolation kit from Miltenyi Biotec (#130-100-629; Auburn, CA, USA). Isolated monocytes were seeded on non–tissue culture-treated plates in α-MEM containing 10% FBS, supplemented with 100 ng/mL M-CSF (in the form of 10% v/v culture supernatant from CMG 14–12 cells) and cultured for 4 days to induce differentiation into bone marrow derived macrophages (BMDM) [ 72 ]. BMDM were used to extract RNA for real-time PCR or for in vitro osteoclastogenesis…”