Inos cxnft

Murine glioma cells (GL261 and GL261-Luc) were a generous gift from Dr. G. Yancey Gillespie (UAB Brain Tumor Model Core Facility) and were grown in DMEM/F12 10% FBS, L-glutamine, and Pen/Strep as described [43 (link)]. Bone marrow derived macrophages (BMDM) were isolated and cultured as previously described [14 (link)]. For immunoblotting experiments, antibodies for phospho-STAT3 (Y705) and total STAT3 were purchased from Cell Signaling (9131; 79D7), and GAPDH from AbCam (ab8245). For IHC, rabbit anti-Iba1 was purchased from Wako (019-19741), rabbit anti-vWf from Millipore (AB7356) and rabbit anti-Ki67 from Abcam (AB15580). For flow cytometry experiments, antibodies for CD45 (30-F11), CD11b (M1/70), Gr-1 (RB6-8C5), F4/80 (BM8), CD4 (GK1.5), CD8 (53-6.7), Foxp3 (MF-14), CD25 (PC61) were purchased from BioLegend, iNOS (CXNFT) from eBioscience, and Arg1 (IC5868A) from R & D Systems [19 (link), 44 (link)]. The mouse cytokine/chemokine multiplex ELISA assay was purchased from EMD Millipore (MPXMCyto-70K).

After LPS stimulation for 0, 4 or 24 h as described above, macrophages were harvested after removal of supernatant and washing with PBS. For flow cytometry, cells were stained with viability dye (Ghost UV450 or Ghost Red710 Tonbo Bioscience, San Diego) using an intracellular staining protocol (IC fixation and permeabilization staining kit, eBioscience). Antibodies included iNOS (CXNFT, eBioscience catalogue no. 12-5920), CD11b (M1/70, eBioscience catalogue no. 25-0112), F4/80 (BM8, eBioscience catalogue no. 48-4801), Arginase-1 (polyclonal, R&D Systems catalogue no. IC5868F). All antibodies were used as 1:200 dilution. Macrophages were analysed by flow cytometry on an LSRII instrument using FACS Diva software (BD Bioscience) and analysed using FlowJo software (Tree Star, San Carlos, CA). The cytometric bead array (BD Bioscience) was used to determine cytokine concentrations in macrophage supernatant after LPS treatment according to manufacturers’ instructions.