Hypoxia and CO2 Modulation in Macrophages

Monocytes are reportedly the main cellular source of proinflammatory cytokines.^{19 (link),20 (link)} Thus, the THP-1 human monocytic cell line was used in the present study; these cells were purchased form American Type Culture Collection (Manassas, VA, USA). THP-1 cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (Invitrogen; cat. no. 16140071) and 1% penicillin-streptomycin solution at 37°C in a 5% CO₂ incubator. THP-1 monocytes were cultured in six-well plates (1 × 10⁶ cells/mL, 2 mL) for 48 hours in the presence of 100 nM phorbol 12-myristate 13-acetate,^{21 (link)} which stimulated differentiation into macrophages. Macrophages were randomly divided into six groups: control (exposed to 5% CO₂ + 20% O₂), hypoxia + 5% CO₂ (exposed to 5% CO₂ + 0.2% O₂), hypoxia + 7% CO₂ (exposed to 7% CO₂ + 0.2% O₂), hypoxia + 10% CO₂ (exposed to 10% CO₂ + 0.2% O₂), hypoxia + 10% CO₂ + N-acetyl-L-cysteine (NAC), and hypoxia + 10% CO₂ + Z-YVAD-FMK. Cells in the hypoxia + 10% CO₂ + NAC and hypoxia + 10% CO₂ + Z-YVAD-FMK groups were respectively treated with an ROS scavenger, 25 mM NAC^{22 (link)–24 (link, link)} (MedChemExpress, Monmouth, NJ, USA; cat. no. HY-B0215), and a pan-caspase inhibitor, 10 μM Z-YVAD-FMK^{25 (link),26 (link)} (ApexBio, Boston, MA, USA; cat. no. A8955), for 30 minutes before exposure to 10% CO₂ + 0.2% O₂.