Recombinant gm csf

Purified LPS was purchased from InvivoGen (San Diego, CA). AT-406 was synthesized as described^{65 (link)}. Flag-M2 antibodies, Flag-M2 beads, necrostatin-1 (Nec-1), cycloheximide (CHX), thapsigargin (THAP), propidium iodide (PI) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) were purchased from Sigma (St. Louis, MO). JNK inhibitor II (SP600125) and DMSO were purchased from Merck Millipore (Billerica, MA). Annexin V-Cy5 was obtained from BD-Biosciences (Franklin Lakes, NJ). Recombinant GM-CSF was purchased from R&D (Minneapolis, MN, USA). z-VAD-FMK was obtained from Bachem (Bubendorf, Switzerland). Recombinant mouse and human TNF were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant mouse IFNβ was purchased from PBL Assay Science (Piscataway, NJ, USA). BV6 was purchased from Selleck Chemicals (Houston, TX). Recombinant human Fas ligand (FLAG-FasL) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). CellTiter-Glo^® Luminescent Cell Viability Assay was purchased from Promega (Fitchburg, Wisconsin). Lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Constructs of p38α MAPK and active MKK3 (MKK3b(Glu¹⁸⁹, Glu¹⁹³)) were gifts of Dr. Jiahuai Han (Xiamen University, China).

Bone marrow cells were isolated from the femurs and tibias of 5-6-week-old female Balb/c mice. The cells were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and immature bone marrow-derived DCs were differentiated with recombinant GM-CSF (10 ng/ml, R&D SYSTEMS, USA) in a humidified 5% CO₂ incubator at 37°C. On days 3, 5, 7, and 9 of culture, floating cells were gently removed, and fresh, warmed medium with mouse recombinant GM-CSF (10 ng/ml) was added. On day 10, nonadherent cells were harvested and used for in vitro experiments.

The human peripheral blood mononuclear cells were obtained from healthy donors, and human dendritic cells were generated as described previously [27 (link)]. The CD14⁺ cells were positively selected, using the miniMACS system with anti-CD14 microbeads. The immature DCs were generated from CD14⁺ monocytes by culturing in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5 ng/mL recombinant GM-CSF (R&D Systems, Minneapolis, MN, USA), 5 ng/mL IL-4 (R&D Systems, Minneapolis, MN, USA), every 3 days for 6 days in a humidified 5% CO₂ incubator. To trigger the maturation of the DCs, immature DCs were incubated with 5 μg/mL lipopolysaccharide (LPS) for an additional 24 h. In these experiments, the 6-MITC, I7457, and I7557 (0, 1, 3.3, and 10 μM) were added at the beginning of the CD14⁺ cell culture, to determine their effects on DC differentiation and maturation.