Mouse granulocyte macrophage colony stimulating factor gm csf

Six- to eight-week-old female wild-type, OT-1 and OT-II mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were approved by the Institutional Animal Care and Use Committee of Merck & Co., Inc., Kenilworth, NJ, USA.
The pharmacologic HPK1 inhibitor Compound 1 was synthesized according to the methods described in Genentech patent application WO 2018183964 A1. Pembrolizumab and IgG4 isotype were generated by Merck & Co., Inc., Kenilworth, NJ, USA. lipopolysaccharide (LPS) (Sigma-Aldrich, St.Louis, MO), forskolin (FSK) (Sigma-Aldrich, St.Louis, MO), PGE₂ (Cayman Chemical, Ann Arbor, MI), ovalbumin (OVA) peptides (OVA_257-264 and OVA_323-339) (Sigma-Aldrich, St.Louis, MO), and 5’-N-ethylcarboxamido adenosine (NECA) (Tocris, Bristol, UK) were prepared and used according to the manufacturer’s instructions. Anti-human CD3 (hCD3) (Clone OKT3), anti-human CD28 (hCD28) (Clone CD28.2), anti-mouse CD3 (mCD3) (Clone 145-2C11), anti-mouse CD28 (mCD28) (Clone 37.51), and carboxyfluorescein succinimidyl ester (CFSE) cell labeling kit were from Thermo Fisher Scientific (Waltham, MA). Mouse granulocyte macrophage-colony stimulating factor (GM-CSF) and mouse IL-4 were obtained from Peprotech (NJ, USA). All flow antibodies were purchased from Biolegend or BD Bioscience (San Diego, CA).

BMDCs were prepared from tibias and femurs of six–eight-week-old healthy female C57BL/6 and BALB/c mice. Bone marrow cells were passed through a 40 μm cell strainer (BD Falcon). After centrifugation at 200 × g for 3 min, red blood cells were lysed with 2 mL of ACK lysis buffer (Biolegend, USA) for 10 min. The remaining cells were then plated in six-well plates at a density of 1×10⁶ cells/mL in RPMI 1640 medium supplemented with 10% FBS, 20 ng/mL mouse granulocyte macrophage colony-stimulating factor (GM-CSF) (Peprotech, USA), 10 ng/mL mouse IL-4 (Peprotech), and 50 μM β-mercaptoethanol (Sigma, USA). Half of the medium was replaced with an equal volume of fresh medium containing the cytokines every three days. Seven days after the initial plating, non- and semi-adherent cells were harvested and regarded as mature BMDCs, and were loaded with mitomycin C treated tumor cells (1:5) or long peptides (20 μg/mL) for two days.
Spleens were harvested under sterile conditions from C57BL/6 and BALB/c mice with local treatment and T cells were isolated as responder cells using a 40 μm cell strainer. Mixing splenocytes with the antigen loaded BMDCs (10:1). After 72–96 h of incubation, cell clusters were observed and imaged under light microscope (CKX31, OLYMPUS, Japan).