Granulocyte macrophage colony stimulating factor gm csf

Murine BC 4T1 cell line was maintained and cultured as our previous reports (7 (link), 13 (link)). Sax (0, 0.2, and 0.4 μM) and Sit (0, 0.6, and 1.2 μM) (MCE, Houston, USA), NRF2 specific inhibitor ML-385 (0, 5, and 10 µM) (MCE, Houston, USA), Heme oxygenase 1 (HO-1) specific inhibitor HO-1-IN-1 hydrochloride (0, 5, and 10 µM) (MCE, Houston, USA), reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) (0, 2.5, and 5 mM) (AbMole, Houston, USA), and NRF2 activator alpha-lipoic acid (ALA) (0, 40, and 60 μM) (Dandong Yichuang Co, China) were used as described previously (7 (link)). pNF-кB-TA-luc reporter plasmids were obtained from Beyotime, Jiangsu, China, as described previously (14 (link)). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from PeproTech, Cranbury, USA. NLRP3 inhibitor MCC950 (0, 5, and 10 µM) (MCE, Houston, USA), caspase-1 inhibitor VX-765 (0, 10, and 20 µM) (AbMole, Houston, USA), and nuclear factor kappa B (NF-κB) specific inhibitor BAY 11-7082 (0, 2, and 4 µM) (Selleck, Houston, USA) were used in this study. For pharmacological intervention assays, 4T1 cells were pretreated with Sax (0.4 μM) or Sit (1.2 μM) for 16 h and then cotreated with indicated inhibitors or activators for additional 4–6 h unless otherwise specified. All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated.

Peripheral blood mononuclear cells (PBMCs) were isolated from leucocyte cones of healthy deidentified blood donor volunteers from the NHS Blood and Transplant bank, and experiments were conducted in accordance with a protocol approved by Queen Mary Research Ethics Committee (QMREC 2014:61) and in accordance with the Helsinki Declaration. PBMCs were isolated following density gradient centrifugation using Histopaque 1077. These were then plated in 10‐cm² plate (Greiner) at a density of 2 × 10⁷ cells/plate, and monocytes were allowed to adhere to the culture plates for 1 h in PBS (Ca²⁺/Mg²⁺). Non‐adhering cells were removed with washes with PBS Ca²⁺ and Mg²⁺ free. Monocyte‐to‐macrophage differentiation was induced for 7 days in RPMI 1640 medium supplemented with 10% foetal bovine serum towards either M1‐differentiated macrophages (M1D; 50 ng/ml granulocyte–macrophage colony‐stimulating factor, GM‐CSF; PeproTech) or M2‐differentiated macrophage (M2D; 50 ng/ml macrophage colony‐stimulating factor, M‐CSF; PeproTech). For macrophage activation, M1D macrophages were stimulated with 10 ng/ml LPS (InvivoGen, UK) and 20 ng/ml IFNγ for 24 h (M1A), whereas M2D macrophages were stimulated with 20 ng/ml IL‐4 for 48 h (M2A) (Martinez et al, 2006; Däbritz et al, 2015; Fig EV1A).

Human blood collected from healthy donors was obtained at the Regional Blood Bank (Lille, France). This study was conducted in accordance with the rules of the Declaration of Helsinki of 1975, revised in 2008. Written consent was obtained from donors by the blood bank, and they were informed that the blood would be used for research purposes.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Ficoll-Hypaque^TM PLUS (GE Healthcare, Vélizy-Villacoublay, France), as described previously [15 (link)]. Cells were resuspended in non-supplemented RPMI 1640 medium, and an average of 10⁷ cells per well (~1.5 million/cm²) was seeded in a Falcon® polystyrene six-well plate (Fischer Scientific, Illkirch-Graffenstaden, France). Monocytes from the PBMCs were allowed to adhere to plastic for 2 h at 37 °C, 5% CO2, and non-adherent cells were subsequently removed by aspiration and extensive washing with PBS. Monocytes were cultivated in serum-free medium (SFM) containing either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, Neuilly-sur-Seine, France) at 20 ng/mL. The medium was changed on Day 3. On Day 7, the cells were differentiated into macrophages. All media were purchased from Gibco® (Thermo Fischer Scientific, Villebon sur Yvette, France).

BMDCs were isolated from the femurs and tibias of C57BL/6 male mice as previously published [14 (link)]. Briefly, the muscle tissue around femurs and tibias of the killed mice was removed, the bone marrow suspension was collected and centrifuged at 12,000× rpm for 5 min, and the supernatant was discarded. The cells were resuspended in red blood cell lysis buffer (BD, Franklin Lakes, NJ, USA), incubated at room temperature for 3–5 min, centrifuged at 12,000× rpm for 5 min, and washed twice with RPMI 1640 medium (Gibco, Grand Island, NY, USA) with 10% FBS. The cells were added to a 6-well culture plate (2 × 10⁶ cells/well), and then interleukin (IL-4) (1000 U/mL) (PeproTech, Cranbury, NJ, USA) and granulocyte-macrophage colony stimulating factor (GM-CSF) (1000 U/mL) (PeproTech, Cranbury, NJ, USA) were added. Every 2 days, the culture plate was gently shaken and 3/4 of the culture medium volume was replaced with fresh culture medium containing IL-4 and GM-CSF. On the eighth day, the cells were collected for subsequent experiments.

Human monocytes were isolated and M1 and M2 macrophages were generated using previously published methods [24 (link)]. In brief, human PBMCs were acquired from the New York Blood Center (New York, NY) from healthy donors aged between 20 and 40. PBMCs were processed with a PBS wash and red blood cell lysis with ACK lysing buffer (118-156-101, Quality Biological). CD14 + monocytes were isolated by magnetic bead separation (17,858, STEMCELL Technologies) and cryopreserved in 95% heat-inactivated fetal bovine serum (HI-FBS) (16,140,071, Gibco) and 5% DMSO (4-X, ATCC).
Monocytes were thawed in RPMI (11,875,119, Gibco) supplemented with 10% HI-FBS and 1% penicillin–streptomycin (11,995,073, Gibco). For M1 macrophages, monocytes were differentiated with 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) (300-03, PeproTech) for five days, followed by M1 polarization with 20 ng/mL GM-CSF, IFNγ (300–02, PeproTech), LPS (L3012, Sigma-Aldrich), and IL-6 (200-06, PeproTech) for four days. M2 macrophages were differentiated with 20 ng/mL macrophage colony-stimulating factor (M-CSF) (300-25, PeproTech) for five days, followed by M2 polarization with 20 ng/mL M-CSF, IL-4 (200-04, Peprotech), IL-13 (200–13, Peprotech), and IL-6.