Gm csf

Bone marrow cells were obtained from the femurs and tibiae of mice by flushing with a 22-gauge needle and then were resuspended. After rinsing by centrifugation, cells were resuspended and counted. A total of 5 × 10⁵ marrow cells were plated into six well plates in 2 mL of culture medium for both the CFU-F and CFU-GM assays. Triplicate cultures were established. After 3 days of adhesion, unattached cells were removed and fresh medium was added to the adherent cells. The whole medium was changed every 3 days. On day 14, colonies were stained with 0.1% crystal violet (Solarbio). Colonies consisting of >50 cells were defined as CFU-F or CFU-GM. The colony- forming efficiency was determined by counting the number of colonies per 5 × 10⁵ marrow cells plated. CFU-Fs were cultured in α-MEM medium (Hyclone; Thermo Scientific, USA) with 2 mmol·L^-1 glutamine (Gibco BRL, Grand Island, USA), 100 U·mL^-1 penicillin (Gibco), 100 U·mL^-1 streptomycin (Gibco) and 20% fetal bovine serum (FBS; Gibco). CFU-GMs were cultured in α-MEM medium with 20 ng·mL^-1 M-CSF (Novus Biological Inc., Littleton, CO, USA), 2 ng·mL^-1 GM-CSF (Novus), 2 mmol·L^-1 glutamine, 100 U·mL^-1 penicillin, 100 U·mL^-1 streptomycin, and 20% FBS.

Monoclonal antibodies (mAbs) to rat CD4, CD8, CD134, CD45, CD11b, TCRαβ, IFN-γ, IL-17A, RT1B (MHC II), CD40, CD45RA, CD62L, CD32, IL-4, secondary reagents and isotype controls were obtained from BD Biosciences Pharmingen (Mountain View, CA, USA). Additional mAbs to rat CD11b (Serotec, Oxford, UK), CD25 and IL-17A (eBioscience, San Diego, CA, USA), TCRαβ and CD43 (BioLegend, San Diego, CA, USA), CCR2 and CCR6 (R&D Systems, Inc., Minneapolis, MN, USA), and GM-CSF (Novus Biologicals, Littleton, CO, USA) were also used. Polyclonal Abs to CX3CR1 and CCR7 were acquired from Abcam (Cambridge, UK).