Rmgm csf

Bone marrow derived macrophages were differentiated with 10 ng/ml rmGMCSF (Peprotech) in RPMI 1640 medium containing 10% (v/v) foetal bovine serum for seven days from cells isolated from femurs of 10–12 week old mice. They were then fixed and stained with Oil red O for 15 minutes at room temperature. Using light microscopy, cells were evaluated blinded for inclusion of Oil Red O lipid droplets.

Bone marrow- derived dendritic cells (BMDC) were prepared according to standard protocol. Briefly, mice were sacrificed and tibiae were removed. Marrow from tibiae was isolated and erythrocytes were lysed. Cells were washed and resuspended in DC medium (RPMI 16–40, 10%FBS, 1% Pen/strep, 50uM 2-ME, L-glutamine 2mM) plus 20ng/ml rmGM-CSF (Peprotech) at a concentration of 2x10⁶ cells per 100mm dish. After 10 days, cells were replated at 1x10⁶/ml for experiments in DC medium without GM-CSF. For the relevant experiments cells were stimulated with 10ng/ml LPS (Alexis Biochemicals) or with freshly prepared primary apoptotic cells at a ratio 1:1.

Bone marrow cells were obtained from C57BL/6 mice and were separated and prepared as single-cell suspensions; erythrocytes were lysed with ACK buffer. The residual bone marrow cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (HyClone), 100 IU/ml penicillin (Gibco), 1% sodium pyruvate (Corning), and 1% HEPES in the presence of 50 ng/ml rm-GM-CSF (Peprotech) and 2.5 ng/ml rm-IL-4 (Peprotech) to induce the differentiation of dendritic cells. After 6 days of incubation at 37 °C and 5% CO₂, the dendritic cells were identified by using FACS staining with specific fluorescence-labeled CD11c antibody (Biolegend). The dendritic cells were incubated with ALD-DNA overnight with or without additional NaCl (20 mM, Sigma)^{31 (link),32 (link),70 (link),101 (link)} and with or without the STAT1 inhibitor fludarabine (2 μg/ml, Selleck-21679-14-1) and p38 MAPK kinase inhibitor (5 μM, InvivoGen-tlrl-sb20). The harvested dendritic cells were collected for transfer to C57BL/6 mice or for flow cytometric measurement of activation and/or maturation markers.

Bone marrow cells were collected from the femurs of BALB/c mice and cultured in RPMI 1640 (GIBCO, Grand Island, NY, USA) medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, GO, Brazil), and antibiotic-antimycotic (GIBCO, Grand Island, NY, USA). BM-DCs differentiation was induced by 20 ng/mL of granulocyte–macrophage colony-stimulating factor (rmGM-CSF, Peprotech, Rocky Hill, NJ, USA). After 6 days, the cells were harvested, pulsed with 100 μg/mL of chicken ovalbumin (OVA grade V; Sigma-Aldrich, Saint Louis, MO, USA), and treated with 50 μM of the PAFR antagonist WEB 208630 (link) (Tocris Biosciences, Ellisville, MO, USA), for 30 min. LPS (1 μg/mL, Sigma-Aldrich), was then added to induce cell maturation. After 24 h, the cells were extensively washed to remove free OVA, and were re-suspended in saline at 10⁶ cells/100 μL. Three to five million cells were transferred to naïve mice via intraperitoneal injection. This process was repeated after 7 days. At day 14 of the first cell transference, the mice were euthanized by CO₂ exposure, and their serum and spleen were collected.

The proliferation capacity of BMDCs was detected using MTT assay (YEASEN). DCs were plated at a density of 4000 cells/well in 96-well plates and cultured in complete RPMI-1640 medium supplemented with rmGM-CSF and IL-4 (PeproTech) for six days. Plates of cells were added with 5 mg/mL MTT solution and dissolved in DMSO 3h later. The absorbance was measured at 490 nm.

Bone marrow dendritic cells (BMDC) were isolated as previously described [49 (link)]. Briefly, the femurs and tibias of euthanized WT mice were removed and placed in cold 70% ethanol for 5 min followed by a 5 min wash in sterile PBS. Then, both ends were cut with the marrow flushed with RPMI using a 0.22 mm syringe into a 70 um mesh. Cells were grown in RPMI supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 10% FCS in addition to 20 ng/mL rmGM-CSF (Peprotech, Cat#315-03). On day 3, another 10 mL RPMI containing 20 ng/mL rmGM-CSF was added to the original culture. On days 6 and 8, half of the culture supernatant was collected, and the cell pellet was resuspended in fresh 10 mL RPMI with 20 ng/mL rmGM-CSF. The purity of the culture was checked by flow cytometry at day 6, which was >70% positive for CD11c and MHC-II, at which time cells were used for downstream in vitro experiments.