Rmgm csf

Primary culture of BMDCs was obtained using mouse recombinant granulocyte-macrophage colony stimulating factor (rmGM-CSF; Sigma-Aldrich, St. Louis, MO, USA), as described previously [45 (link)] with minor modifications. Bone marrow isolated from the tibia and femur was suspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 1% antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin, Symbios, Gdansk, Poland). Erythrocytes were lysed with NH₄Cl-Tris buffer. Cells were resuspended in R-10 growth medium [RPMI-1640 medium enriched with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT, USA), 1% antibiotic solution (100 U/mL penicillin and 100 μg/mL streptomycin, Symbios), 50 μM 2-mercaptoethanol (Sigma-Aldrich), and 20 ng/mL rmGM-CSF], and then placed into wells of a six-well plate and incubated at 37 °C in the presence of 5% CO₂. The growth medium was added sequentially on day 3 of culture and replaced partially on day 6 of culture. On day 8 of culture, BMDCs were enriched using MACS CD11c⁺-labeled magnetic beads (Miltenyi Biotec, Auburn, CA, USA). Cell viability was above 95%, as determined by the 0.4% trypan blue exclusion test.

C57BL6 BM-derived immature DCs (imDCs) were generated as described previously [16 (link), 17 (link), 29 (link)]. Briefly, BM was harvested from the femurs and tibiae of mice and cultured in RPMI-1640 medium (Gibco-BRL) supplemented with 10% (v/v) FBS (Gibco-BRL) and 1% (w/v) PS in the presence of 20 ng/mL recombinant murine (rm) GM-CSF (R&D Systems, Minneapolis, MN, USA) and 10 ng/mL rmIL-4 (R&D Systems). On culture days 2 and 4, half of the medium was removed and replaced with fresh media containing cytokines. On day 6, imDCs were purified via positive selection with CD11c⁺-magnetic beads (Miltenyi Biotec Inc., Auburn, CA, USA). Then, mature DCs (mDCs) were generated by further cultivation for 48 h of CD11c⁺ DCs with 1 μg/mL lipopolysaccharide (LPS) from Escherichia coli (Sigma-Aldrich) and 10 ng/mL rmGM-CSF.