Recombinant human granulocyte macrophage colony stimulating factor rhgm csf

HLA-A2⁺ human PBMC were cultured in RPMI-1640 medium supplemented with 10% FBS, 500 IU/mL rhIL2, 20 ng/mL recombinant human Interleukin 4 (rhIL4) (Miltenyi Biotec), and 20 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (Miltenyi Biotec) for 7 days for DC cell expansion. The PBMC with enriched DC cells were primed with 100 ng/ml MHSP65 protein Biovison, USA) and 50 µg/ml TNBC cell lysate (TCL) for further DC expansion and activation. After 3 days of TCL priming, the DC cells were ready to use for T cell stimulation. TCL was prepared through the freeze-and-thaw cycle of TNBC cell suspension in PBS buffer.

Synthetic M-triDAP, ultrapure LPS from E.coli O111:B4 and synthetic polyriboinosinic:polyribocytidilic acid (poly-I:C) were from In vivogen (San Diego, CA). LPS was confirmed to be free of NOD1/NOD2 agonists using a test system described earlier (29 (link)). All agonists were dissolved in endotoxin-free water (In vivogen). Complexes of poly-I:C with Lipofectamine 3000 (lipo-poly-I:C) were prepared as recommended by the manufacturer of Lipofectamine (Thermo Fisher Scientific, Waltham, MA). Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was from Miltenyi Biotec (Bergisch Gladbach, Germany), and recombinant human interferon-beta (rhIFN-β1b) from Generium (Moscow, Russia). Complete culture medium (CCM) was RPMI-1640 supplemented with 2-mM L-glutamine and 10% fetal calf serum (all from Thermo Fisher Scientific). IκB kinase β inhibitor (PF-184) was from Tocris (Bristol, UK), actinomycin D (ActD) from Sigma (St-Louis, MO).