Anti cd3

Splenocytes were isolated from dissected spleen of adult (6 to 10 weeks old) male BALB/c mice (Jackson Laboratory, Bar Harbor, ME) euthanized with CO_2. For T-cell activation, the splenocytes (2.5 × 10⁵ cells/well) were incubated with RMPI 1640 medium (Gibco) containing 5% FBS in the 96-well plates coated with anti-CD3 (Corning) [23 (link)] for 6 or 18 hours (hrs) with or without EVs. For LPS stimulation, splenocytes (5 × 10⁵ cells/well; 96 wells) were incubated with RMPI 1640 medium containing 5% FBS and 50 ng/ml LPS (Sigma, Saint Louis, MO) for 4 or 18 hrs with or without EVs. After stimulation, cell-free conditioned media were harvested to measure cytokine levels.

After 30 days of treatment, the lymph nodes were removed and macerated. Cell suspensions were washed and re-suspended in complete RPMI-1640 (Sigma; supplemented with 5% of fetal bovine serum, 40 μg/ml of gentamicin, 20 mM Hepes).
In these tests, preparations of whole cell from organs were used. Cell suspensions containing 5 × 10⁶ cells in 200 μl were incubated in the presence or absence of OVA grade III – Sigma (200 μg/ml) or anti-CD3 (10 μl/ml) in 96 well, flat bottom (Costar 3595, Coming, NY, USA). Cells were cultured for 72 h at 37°C in an oven containing 5% CO₂, and 18 h before the end of the culture, cells were incubated with [³ H] thymidine at a concentration of 0.25 μCi/well. Cells were collected in cells collector (Inotech cell Harvester, USA). Reading was held in a β-scintillation counter (Beckman, Brea, CA, USA). Posteriorly, the supernatants were collected and frozen at 20°C for subsequent measurement of the cytokines IL-5 and IFN-γ by ELISA.