Arginine free rpmi

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors by density gradient centrifugation on FicollPaque PLUS (GE Healthcare-Amersham, NJ, USA), as previously described 19 . Brie y, PBMCs were stained with 0.5 µM CellTrace™ Violet Cell Proliferation Kit (Invitrogen, Molecular Probes, MA, USA), according to manufacturer's instructions. CellTrace-labelled PBMCs were activated with coated 1 µg/ml anti-CD3 and 5 µg/ml soluble anti-CD28 (BioLegend) for 4 days and co-cultured in at bottom 96 well plates at the 1:1 ratio with CD11b -, CD11b + and unsorted fractions. Cell cultures were incubated at 37°C and 5% CO 2 in arginine free-RPMI (Biological Industries), supplemented with 150 µM arginine, 10% FBS (Sigma-Aldrich), 10 U/ml penicillin and streptomycin, and 10 mM HEPES. After 4 days, cells were harvested, stained with anti-CD3 (Beckman Coulter) and analyzed by ow cytometry. Proliferation of T cells was evaluated by assessing the signal of CellTrace on CD3 + cells, and considering as proliferating the cells present from generation 2 onwards, or by calculating the absolute number of CD3 + /CellTrace + cells in each sample by BD TruCount™ tubes (BD Biosciences). In both cases data were normalized assuming the proliferation of T cells cultured alone as 100%.

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors by density gradient centrifugation on FicollPaque PLUS (GE Healthcare-Amersham, NJ, USA), as previously described 19 . Brie y, PBMCs were stained with 0.5 µM CellTrace™ Violet Cell Proliferation Kit (Invitrogen, Molecular Probes, MA, USA), according to manufacturer's instructions. CellTrace-labelled PBMCs were activated with coated 1 µg/ml anti-CD3 and 5 µg/ml soluble anti-CD28 (BioLegend) for 4 days and co-cultured in at bottom 96 well plates at the 1:1 ratio with CD11b -, CD11b + and unsorted fractions. Cell cultures were incubated at 37°C and 5% CO 2 in arginine free-RPMI (Biological Industries), supplemented with 150 µM arginine, 10% FBS (Sigma-Aldrich), 10 U/ml penicillin and streptomycin, and 10 mM HEPES. After 4 days, cells were harvested, stained with anti-CD3 (Beckman Coulter) and analyzed by ow cytometry. Proliferation of T cells was evaluated by assessing the signal of CellTrace on CD3 + cells, and considering as proliferating the cells present from generation 2 onwards, or by calculating the absolute number of CD3 + /CellTrace + cells in each sample by BD TruCount™ tubes (BD Biosciences). In both cases data were normalized assuming the proliferation of T cells cultured alone as 100%.