Fetal calf serum fcs 10

CD4⁺CD25⁺ (nTregs) and CD4⁺CD25⁻ T cells from spleens of naive C57BL/6 (WT) and CD8-deficient (CD8^−/−) mice were isolated by collagenase digestion and enriched using nylon wool columns as described previously [13 (link)]. Lymphocytes were further purified by CD4⁺CD25⁺ regulatory T cell MACS beads (Miltenyi Biotec, Bergisch-Gladbach, Germany), resulting in a purity of >95% of CD4⁺CD25⁺ cells. Cells were washed, counted, and resuspended to a final concentration of 14 × 10⁶ cells per mL in complete RPMI 1640 (Mediatech Cellgro, Manassas, VA, USA) tissue culture medium, containing heat-inactivated fetal calf serum (FCS 10%; Sigma-Aldrich, St. Louis, MO, USA), L-glutamine (5 mM), β-mercaptoethanol (2 mM), hepes buffer (15 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) (all from Gibco, Grand Island, NY). Cells were cultured in media or in the presence of PMA (50 ng/mL) and ionomycin (1 μM, both Sigma-Aldrich, St. Louis, MO, USA) for 3 h.

To determine immune responses against lung elastin, spleen mononuclear cells (MNCs) from PPE-treated mice on day 21 were isolated by Histopaque 1083 (Sigma-Aldrich, St. Louis, MO) as described previously [26 (link)]. Cells were washed, counted, and suspended in complete RPMI 1640 tissue culture medium (Mediatech Celgro, Manassas, VA), containing heat-inactivated fetal calf serum (FCS 10%; Sigma), L-glutamine (5 mM), α-mercaptoethanol (2 mM), Hepes buffer (15 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) (all from Invitrogen). Cells (8 × 10⁶/mL) were cultured with 2, 10, or 50 µg/mL mouse lung elastin peptide (EP, Elastin Products Company) for 4 days and IFNγ and IL-17 levels in culture supernatants were measured by ELISA (eBioscience, San Diego, CA).