Gm csf

BMDCs were generated from bone marrow precursors of C57BL/6 WT, flushed from femurs, in Iscove’s modified Dulbecco’s medium (IMDM) (Euroclone) containing 10% heat-inactivated fetal bovine serum (Euroclone), 100 IU of penicillin, streptomycin (100μgmL⁻¹), 2 mM l-glutamine (Euroclone), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 to 20ngmL^–1) for 8 days.

PBMCs were isolated from buffy coats of healthy donors by density gradient centrifugation using Lympholyte-H (Cederlane Laboratories). In this study we used anonymous residual samples (buffy coats) and approval was not necessary. These residual samples were collected in the Azienda Ospedaliera Universitaria Policlinico Umberto I - Centro Trasfusionale e Immunoematologia. URL: www.policlinicoumberto1.it.
Monocytes were positively separated by anti-CD14 magnetic beads (MACS; Miltenyi Biotec) according to the manufacturer’s instructions. The cells were then resuspended in RPMI 1640 (Euroclone) supplemented with 10% FCS (HyClone, Invitrogen Life Technologies), L-glutamine (2 mM), HEPES buffer (10 mM), and gentamicin (10 µg/ml) (Sigma-Aldrich), and cultured for 5 days in the presence of GM-CSF (200 U/ml) and IL-4 (10 ηg/ml; Euroclone) to generate immature DC (imDC). Then, imDC were put in culture with different concentration of plant extracts in the presence of 200 ηg of LPS.