Humidified c chamber

Treated 24-well plates were incubated at 37 °C for 2 hours with PBS containing 0.1 μg/mL anti-CD3 clone OKT3 (Biolegend, San Diego, CA, USA) and 0.1 μg/mL anti-CD28 clone CD28.2 (Thermo Fisher). PBMC were suspended at 1 × 10⁶ cells/mL in AIM V medium containing 10% FBS and 10 ng/ml of IL-2 (Thermo Fisher). The wells were rinsed with PBS, then 0.5 mL of PBMC were added per well. The next day, 20 uL of lentivirus was added to the cells, along with 5 μg/mL of DEAE-dextran (Sigma, St. Louis, MO, USA). The day after that (day 2), another 20 ul of lentivirus was added to the cells. Three days later, half of the cells were transferred into a humidified C chamber (Biospherix, Parish, NY, USA) set to 5% carbon dioxide and either 5% oxygen or 1% oxygen. As the T cells proliferated over the next 8 days, the cells in the normal incubator and hypoxia chamber were counted every 2–3 days and fresh medium (equilibrated overnight in the hypoxia chamber for the hypoxic cultures) with IL-2 was added to the cultures to maintain the proper cell density.

ATII and A549 cells were exposed to 40 (normocapnia, Ctrl) or 120 mmHg (hypercapnia, CO₂) CO₂ conditions unless otherwise indicated. Normocapnia and hypercapnia media solutions were prepared freshly with DMEM or DMEM/F12, Ham’s F12, and MOPS base to obtain a final pH of 7.4 at 40 (5%) or 120 mmHg (15%) of CO₂ while maintaining 21% O₂ balanced with N₂ and were kept overnight in the humidified C-chamber (BioSpherix Ltd., Parish, NY, USA), as described previously [24 (link)]. Levels of CO₂ in the chamber were controlled by an RO-CO₂ carbon dioxide controller (BioSpherix Ltd., NY, USA). Before experiments media pH, pCO₂ and pO₂ levels were measured using a Rapid-lab blood gas analyzer (Siemens, Erlangen, Germany).