Anti cd3ε antibody clone 145 2c11

FluoroSpot assays to determine frequencies of cytokine secreting cells were performed according to standard procedures and as previously described using commercial available antibody sets for mouse IFNγ, IL-17, and IL-4 (Maptech, San Diego, CA, USA) (Lambracht-Washington et al., 2011 (link); Lambracht-Washington and Rosenberg, 2015 (link)). 2.5 × 10⁵ splenocytes were added per well and were cultured in medium only, or re-stimulated with Aβ42 peptides (10 μg/ml), and incubated at 37 °C in a 5% CO2 humidified incubator for 48 h. For maximal T cell stimulation an anti-CD3ε antibody (clone 145–2C11, eBioscience, Tonbo) was used at 0.5 μg/ml in the cell cultures. Spots were counted by a FluoroSpot plate reader service (Zellnet consulting, Fort Lee, NJ).

A single-cell suspension was prepared from arthritic paws as follows. Each arthritic paw was digested in 1 ml of deoxyribonuclease I (0.1 mg/ml) and liberase (0.313 mg/ml) (both from Roche Diagnostics) for 90 min. Cells (2 × 10⁵ per well) were cultured in a 96-well plate in 200 μl of RPMI 1640 with l-glutamine, 10% fetal calf serum, and penicillin/streptomycin (all from Life Technologies). To detect cytokine secretion, paw cells were stimulated with an anti-CD3ε antibody (clone 145-2C11, eBioscience) for 48 hours.
For measurement of proliferative responses, inguinal lymph node and arthritic paw cells were cultured for 48 hours in the presence of anti-CD3ε (0.1 μg/ml; clone 145-2C11) (eBioscience). The thymidine analog 5-bromo-2′-deoxyuridine (BrdU; 50 μM) was added for 18 hours to access cell proliferation. BrdU staining was carried out according to the manufacturer’s protocols with an anti-BrdU fluorescein isothiocyanate–conjugated antibody (clone B44, BD Biosciences).