Isolation and Coculture of Mouse CD4+ T Cells

Magnetic bead separation (130-049-201, Miltenyi Biotec, Germany) were utilized to isolate mice spleen CD4⁺ T cells from untreated healthy mice using the protocol reported in our previous studies 14 (link), 25 (link). BECs were pretreated with 1 nM of E2, 20 nM of DHT and 20:1 nM of DHT:E2 in the asthma+E2, asthma+DHT, asthma+DHT+E2 groups for 24h, respectively. Then BECs were cocultured with CD4+ T cells at a ratio of 10: 1 (TCs: BECs) 13 for 24h with complete RPMI 1640 culture medium (Gibco, Australia) supplemented with 10% FBS, 1% penicillin and streptomycin, soluble anti-CD28 (1.0 μg/ml, eBioscience), soluble anti-CD3 (0.5 μg/ml, eBioscience), and IL-2 (20 ng/ml, eBioscience) in transwell plate (Corning, USA). After 24h, cells were collected and then detected by flow cytometry.