Murine granulocyte macrophage colony stimulating factor gm csf

GMS and Nor were obtained from Aladdin, Shanghai, China. Poloxamer 188 was obtained from Beijing Probe Bioscience Co. Ltd., Beijing, China. Bicinchoninic acid assay kit, SDS–polyacrylamide gel electrophoresis (PAGE) kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, DiI, and DiO were obtained from Beyotime Biotechnology, Shanghai, China. Mueller-Hinton broth (MHB) and agar were obtained from BD Difco, USA. RPMI 1640 cell culture medium was obtained from Gibco, Thermo Fisher Scientific, USA. IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from NeoBioscience Co. Ltd., Shenzhen, China. LIVE/DEAD BacLight bacterial viability kit (L-7012) was bought from Invitrogen, Thermo Fisher Scientific, USA. Fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD11c, phycoerythrin (PE)–conjugated anti-mouse CD80, PE-conjugated anti-mouse CD86, allophycocyanin (APC)–conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8a, and FITC-conjugated anti-mouse CD4 were purchased from Tonbo Biosciences, USA. Murine IL-4, murine granulocyte-macrophage colony-stimulating factor (GM-CSF), and human TNF-α were bought from PeproTech, USA. E. coli (ATCC no. 25922), S. aureus (ATCC no. 6538), K. pneumoniae (ATCC no. 4352), and HL-60 (ATCC no. CCL-240) were obtained from the American Type Culture Collection (ATCC).

Bone marrow-derived progenitor cells were isolated from femurs, tibiae and hip bones of non-transgenic C57BL/6J mice according to established protocols (30 (link)). Progenitor cells were cultivated in RPMI-1640 with 10% (v/v) FBS (heat-inactivated), GlutaMAX supplement (1:100 from stock, Gibco, Thermo Scientific), 50 μM β-mercaptoethanol (Sigma) and 200 U/ml murine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF, PeproTech) at 0.2 Mio. cells/ml. After 9 days of incubation at 37°C and 5% CO₂, progenitor cells completely differentiated into immature bone marrow-derived dendritic cells (BM-DCs).

Bone marrow stem cell progenitors were obtained from the femurs and tibiae of naïve BALB/c mice (n = 3) and cultured in complete medium (CM) consisting of RPMI (1640 with L-glutamine, Lonza, Basel, Switzerland) supplemented with 10% FBS; a mixture of antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, Lonza); and 10 mM HEPES (Lonza) in the presence of 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, London, UK), as previously described [54 (link)]. Fresh medium containing GM-CSF was added to the cultures every 3 days. On day 7, half of the volume was removed, and cells were centrifuged at 500× g for 10 min. The pellet was then resuspended on CM with freshly added growth factor and cultured at 37 °C with 5% CO₂. On day 10, nonadherent cells were collected and considered BMDCs based on the expression of CD11c as described before [20 (link)].