Recombinant mouse gm csf

Bone marrow-derived DCs (bmDCs) were generated as described previously [33 (link)]. Briefly, 2 × 10⁶ bone marrow cells isolated from femurs and tibiae of female C57BL/6 wt, FcγRI^−/−, FcγRII^−/− and FcγRIII^−/− mice were cultivated in 10 mL of RPMI 1640 supplemented with 10% FCS, 2 mM l-glutamine, 500 nM 2-ME and 4 ng/mL of recombinant mouse GM-CSF and recombinant mouse IL-4 (BD Pharmingen, San Jose, CA, USA) for 3 days. A 10 mL portion of fresh medium supplemented with GM-CSF and IL-4 was then added and cells were cultured for another 3 days. On day 6, non-adherent cells were collected, washed and cultivated in fresh 20 mL medium supplemented with GM-CSF and IL-4. On day 8, loosely adherent differentiated bmDCs were harvested and used for experiments. The phenotype of differentiated bmDC cultures was routinely checked by FACS referring to a myeloid DC phenotype (>85% CD11c and >95% CD11b) in bmDC cultures.

M2-10B4 cell line was purchased from ATCC. M2-10B4 cells were cultured in growth media (RPMI-1640 medium with L-glutamine (Mediatech/Cellgro, reference #: 10-040-CV), supplemented with 10% FBS and 100 units/ml Penicillin, 100 μg/ml Streptomycin) at 37°C with 5% carbon dioxide. The bone marrow from femurs and tibias of C57BL/6 mice was used to prepare bone marrow macrophages and dendritic cells. The bone marrow derived macrophages were prepared and cultured as previously described [99 (link)]. Dendritic cells were cultured in growth medium including RPMI-1640 medium with L-glutamine supplemented with 10% FBS, 100 units/ml Penicillin, 100 μg/ml Streptomycin, 5ml MEM non-essential Amino Acid solution, 5ml HEPES and 10 ng/ml recombinant mouse GM-CSF (BD Biosciences) as previously described [100 (link)].

Bone marrow‐derived DCs (bmDC) were generated in the presence of recombinant mouse GM‐CSF (4 ng/ml, BD Bioscience, San Jose, CA) and IL‐4 (4 ng/ml, BD Bioscience) as described previously.23 At Day 7, DCs were matured by overnight incubation with 2 μg/ml CpG ODN 1826 (InvivoGen, San Diego, CA). On Day 8, CpG‐matured bmDCs were pulsed with 10 μM chicken ovalbumin (OVA)‐derived SIINFEKL peptide (InvivoGen) for 3 hr, subsequently, cells were washed in PBS (Lonza, Switzerland) and a total of 2 × 10⁵ cells in 50 μl were injected partly intratumorally and peritumorally (i.t./p.t.) or 2 × 10⁵ cells in 100 μl i.v. into the mice and referred as DCVacc or sysDCVacc, respectively.