Human PBMC were isolated from LRS chambers (Stanford Blood Center) and cryopreserved until time of use. CD25+ YT-1 cells (Kuziel et al., 1993 (link)) and PBMC were maintained at 37°C in a 5% CO2 humidified chamber, and cultured in complete RPMI medium (RPMIc) containing 10% FBS and supplemented with 25mM HEPES, 2mM pyruvate, 4mM GlutaMAX, non-essential amino acids, and penicillin-streptomycin (all cell culture reagents were purchased from Gibco). Prior to stimulation for pERK and pAkt studies, cells were starved in serum-free RPMI for 1–2 hours. Primary cells were rested overnight without cytokine before measuring signaling.
Normal human primary bronchial/tracheal epithelial cells were purchased from ATCC (PCS-300–010) and grown in Airway Epithelial Cell Basal Media (ATCC PCS-300–030) supplemented with Bronchial/Tracheal Epithelial Cell Growth Kit components (ATCC PCS300–040) following manufacturer’s instructions. A549 cells were maintained in complete DMEM medium containing 10% FBS and supplemented with 25mM HEPES, 2mM sodium pyruvate, 4mM GlutaMAX, and penicillin-streptomycin. NKL cells were cultured in RPMIc containing 100IU human IL-2, with media and IL-2 changes every other day. All cell lines were maintained at 37°C in a 5% CO2 humidified incubator.
Normal human primary bronchial/tracheal epithelial cells were purchased from ATCC (PCS-300–010) and grown in Airway Epithelial Cell Basal Media (ATCC PCS-300–030) supplemented with Bronchial/Tracheal Epithelial Cell Growth Kit components (ATCC PCS300–040) following manufacturer’s instructions. A549 cells were maintained in complete DMEM medium containing 10% FBS and supplemented with 25mM HEPES, 2mM sodium pyruvate, 4mM GlutaMAX, and penicillin-streptomycin. NKL cells were cultured in RPMIc containing 100IU human IL-2, with media and IL-2 changes every other day. All cell lines were maintained at 37°C in a 5% CO2 humidified incubator.