Anti cd3 and anti cd28 antibodies mouse anti human

To evaluate cell proliferation, 1 × 10⁷ PBMC in 10 ml of PBS were labeled with 5,6-carboxyfluorescein succinimidyl ester (CFSE, 0.5 μM, Invitrogen, Eugene, USA) according to the manufacturer's instructions. 2 × 10⁶ cells/well were seeded in 6 well microtiter plates (TPP, Trasadingen, Switzerland) in 5 ml of 786-0^VHL− or 786-0^VHL+ conditioned media or in co-culture with 3 × 10⁵ 786-0^VHL− / 786-0^VHL+ cells/well in the presence of conditioned media followed by direct stimulation with plate-bound anti-CD3 and anti-CD28 antibodies (mouse anti-human, 1 μg/ml each, BD Biosciences) and maintained for 5 days in culture. 10,000 events were analyzed on a BD FACSCanto II flow cytometer in combination with the FACSDiva software package (BD Biosciences) in a time kinetic. The results were expressed as percentage of proliferating cells.
Proliferation assays in the presence of MnSOD2 were analyzed as described above in the absence and presence of 1U/ml recombinant MnSOD2 (Abnova; Pforzheim, Germany) within the culture medium for up to 40 hours.

To evaluate the VHL-mediated apoptosis induction, 2 × 10⁶ PBMC were cultured in 5 ml of 786-0^VHL−or 786-0^VHL+ conditioned media or in co-culture with 3 × 10⁵ 786-0^VHL− / 786-0^VHL+ cells/well in the presence of conditioned media followed by direct stimulation with plate-bound anti CD3-and anti-CD28 antibodies (mouse anti-human, 1 μg/ml each, BD Biosciences) for 72 h. Apoptosis was determined by flow cytometry after staining of cells with APC-annexin V (BD Phamingen™) and propidium iodide (2 mg/ml, Sigma-Aldrich) according to the manufacturer 's instructions (BD Phamingen™). The stained cells were analyzed using the BD FACSCanto II flow cytometer and the FACSDiva software package (BD).

IL-2 secretion was analyzed using commercial enzyme-linked immunosorbent (ELISA) assay (eBioscience, Austria) based on the supplier's protocol. Therefore, “day 6” T cells were plated at a density of 1 × 10⁵ cells/well onto 96-well culture plates in 200μl of RPMI-1640 (Sigma-Aldrich) supplemented with 10% FCS (PAA) and 2 mM L-glutamine (Lonza) in the absence and presence of MnSOD2 (1U/ml, Abnova via Biozol, Eiching, Germany). T cells were directly stimulated with plate-bound anti-CD3 and anti-CD28 antibodies (mouse anti-human, 1 μg/ml each, BD) in a time kinetic fashion (20 h, 40 h). Next, the supernatants were cleared by centrifugation and measurements were performed according the manufacturer's instructions using 100 μl cell culture supernatants.