Recombinant human m csf

The following antibodies (Abs) and reagents were used in present studies: anti-IL-1β (human specific, mouse specific, and human & mouse specific), anti-phospho NF-κB, anti-NF-κB, anti-phospho ERK, anti-ERK, anti-phospho p38, anti-p38, anti-phospho 4E-BP1, anti-4E-BP1, anti-phospho Mnk1, anti-Mnk1, anti-phospho eIF4E, anti-eIF4E, anti-phospho MK2, anti-MK2, and anti-streptavidin-HRP antibodies were purchased from cell signaling technology (Danvers, MA). Anti-β-actin, Dimethyl sulfoxide, Actinomycin D, Cyclohexamide, ultrapure E. coli O111:B4 LPS, Rapamycin, PD98059, and SB202190 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-NLRP3 antibody was purchased from Adipogen (San Diego, CA). Torin 1 and FR180204 were purchased from Merck Millipore (Billerica, MA). CGP57380 and MK25 were purchased from TOCRIS (Ellisville, MO, USA) and Cayman Chemical (Ann Arbor, MI), respectively. Recombinant human M-CSF and mouse M-CSF were purchased from R&D system (Minneapolis, MN).

Macrophage differentiation was carried out as previously described [4 (link)]. Briefly, PBMC obtained from healthy donors buffy coats were washed with sterile pyrogen-free saline, centrifuged at 100 g for 10 min, and resuspended in AIM-V medium. Fifty ×10⁶ cells were seeded in 75 cm2 culture flasks and cultured for 1 h at 37°C and 5% CO2. Non-adherent cells were removed by washing twice with warm saline, and remaining cells were cultured in AIM-V for 6 days with the addition of 30 ng/ml recombinant human M-CSF (R&D Systems, Minneapolis, MN, USA), to stimulate their differentiation to macrophages (MΦ). Obtained cells were gently resuspended in AIM-V, seeded in 12-well culture plates (5×105 cells/ well) and cultured for 24 h in the presence of 20 μM Gb3 (Matreya, Pleasant Gap, PA, USA) and/or 200 μM 1-deoxygalactonojirimycin (DGJ, Sigma). For dose response experiments, 24 h culture media was replenished with fresh media containing Gb3, DGJ and 2, 5 or 10 μg/ml of PPS and 72 h followed by harvesting of the supernatant. In the case of kinetics experiments, after the initial 24 h media was replenished with fresh media containing Gb3, DGJ and 5 μg/ml of PPS and the supernatant was harvested 24, 48 or 72 h later.

Monocytes were plated onto tissue culture-treated plates at a density of 1.5×10⁵ cells/cm². Monocytes were maintained in MDM medium (.375×10⁶/mL), consisting of X-VIVO™15 (Lonza) or of RPMI 1640 (PAA) with 10% FCS (PAA). The medium was supplemented with 100 ng/mL (approx. 1.7×10⁴ units/mL) recombinant human M-CSF (R&D Systems), as well as, 2 mM glutamine (PAA), 100 U/mL penicillin and 100 µg/mL streptomycin (PAA). Monocytes were incubated at 37°C, with 5% CO₂ and differentiated for 5–7 days prior to use. For activation of macrophages for cytokine profile experiments, IFN-gamma (R&D, 100 U/mL) and LPS (Sigma, 100 ng/mL) were added to the culture medium during the last 16 hours of culture. For MDM analysis, cells were detached using cold PBS containing EDTA (5 mM).

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of healthy volunteers by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich). The cells were washed three times with sterile PBS and resuspended in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin (complete medium). The PBMCs were incubated at 37 °C in complete medium and allowed to adhere for 45 min. The nonadherent cells were removed and the adherent cells were washed with sterile PBS and harvested with a rubber policeman. The purity of the monocytes isolated by Ficoll-Hypaque as determined by flow cytometry was approximately 85–90%. The cells were cultured for three weeks in conditioned mediums from THLE-2 cells in the presence of 25 ng mL⁻¹ recombinant human M-CSF (R&D Systems). The medium was changed every other day. On day 21, TRAP-positive cells were identified using a leukocyte acid phosphatase kit (Sigma-Aldrich)^{10 (link)}.