C chamber

OPCs were plated in OPC growth media and then placed into a 2 shelf C-Chamber from BioSpherix (C-274). Oxygen tension was controlled using the ProOx 110 from BioSpherix such that nitrogen gas would flush out oxygen to maintain the chamber at the desired oxygen level. The subchamber was set at 1% O₂ and cells were cultured for 48 hours unless otherwise noted. Hypoxia treated cells were then rapidly lysed for RNA or protein to minimize degradation of HIFs upon exposure to room air. The normoxic controls were cultured concurrently in the same cell culture incubator containing the BioSpherix C-Chamber.

For the different experimental conditions, initial solutions were prepared with DMEM/Ham’s F-12 medium/tris base/Mops base (3:1:0.25:0.25) containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), as described elsewhere (6 (link)). The buffering capacity of the medium was modified by changing its initial pH with tris and Mops base to obtain a pH of 7.4 at the various CO₂ concentrations (pCO₂ of 5, 7.5, 10, and 20% for 30 to 40, 50 to 55, 60 to 80, and ~120 mmHg, respectively). In some experiments modeling extracellular acidosis, an initial pH of 6.8 was used, resulting in a final pH of 7.2 and a pCO₂ of 40 mmHg. The desired CO₂ and pH values were achieved by equilibrating the medium overnight in a humidified chamber (C-Chamber, BioSpherix). The atmosphere of the C-Chamber was controlled with a PRO CO₂ carbon dioxide controller (BioSpherix). In this chamber, cells were exposed to the desired pCO₂ while maintaining 21% O₂ balanced with N₂. Before and after CO₂ exposure, pH, pCO₂, and pO₂ values in the medium were measured using a Stat Profile pHOx blood gas analyzer (Nova Biomedical). Experiments were started by replacing the culture medium with the CO₂-equilibrated medium and incubating in the C-Chamber for the desired time.