Recombinant human ctla4 ig hum hum

Human umbilical vein endothelial cells were cultured in Medium 199 supplemented with 20% (v/v) fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM l-glutamine, 1 µg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), and 10 µg/mL endothelial cell growth factor supplement (Sigma-Aldrich, St. Louis, MO, USA). Cells from passage 1 or 4 were used in all experiments. Cells grown to confluence were pretreated with INF-γ (400 U/mL) at 37°C for 24 h. After three washes with PBS, the cells were incubated with medium alone, recombinant human CTLA4-Ig (hum/hum) or recombinant human IgG1-Fc isotype control (both from BioXCell, Lebanon, NH, USA) for an additional 24 h. Unstimulated HUVECs were used as controls. The supernatants were collected for the determination of IDO activity by HPLC.

Commercial human aortic SMCs (Cascade Biologics, Life Technologies, CA, USA) were maintained in SMC medium (Lonza, Basel, Switzerland). After 24 h of pre-stimulation with human recombinant IFNγ (400 U/mL), the cells were washed three times with PBS and incubated with medium alone, recombinant human CTLA4-Ig (hum/hum) or recombinant human IgG1-Fc isotype control (both from BioXCell, Lebanon, NH, USA) for 24 h. Unstimulated SMCs were used as controls. The supernatants were collected for the determination of IDO activity by HPLC.

Peripheral blood was obtained from healthy volunteers at the Blood Central of Karolinska University Hospital, Stockholm, Sweden. Peripheral blood mononuclear cells were isolated using Lymphoprep™ gradient medium (density 1.077 g/ml; Axis-Shield, Oslo, Norway) according to the manufacturer’s instructions. After a 1 h adherence step, the floating cells were discarded. The adherent monocytes were used to generate “M0” macrophages as previously described (20 (link)). Briefly, the monocytes were cultured for 6 days in medium [RPMI 1640, 50 U/mL penicillin, 50 g/mL streptomycin (Gibco Invitrogen, Carlsbad, CA, USA), and 10% fetal bovine serum] supplemented with 20 ng/mL M-CSF (R&D Systems, MN, USA). After 24 h of pre-stimulation with human recombinant IFNγ (400 U/mL), the cells were washed three times with PBS and incubated with medium alone, recombinant human CTLA4-Ig (hum/hum) or recombinant human IgG1-Fc isotype control (both from BioXCell, Lebanon, NH, USA) for 24 h. Unstimulated macrophages were used as controls. The supernatants were collected for the determination of IDO activity by HPLC.