Proox p110 oxygen controller

Zebrafish embryos were obtained from adults of the corresponding transgenic lines; transgenic fishes were outcrossed with wild type individuals to ensure fluorescent signal homogeneity in heterozygous offspring. Fluorescent embryos at 24 h post fertilization (hpf) were incubated for 2–5 days at 28°C into a hypoxic chamber (ProOx P110 Oxygen Controller, BioSpherix, Parish, United States) with controlled and constant 5% oxygen concentration (about 2 mg/L, corresponding to a moderate hypoxia). As controls, fluorescent sibs were kept at 28°C outside the hypoxic chamber, under normoxic conditions.
A set of hypoxia treatments was performed following the incubation times applied by Kajimura and colleagues (Kajimura et al., 2005 (link)) to re-check the expression of zebrafish igfbp1a and igfbp1b genes in hypoxic (oxygen at 2 mg/L) and normoxic conditions: 24 h of hypoxia, starting from 1 hpf; 24 h of hypoxia, starting from 24 hpf; 24 h of hypoxia, starting from 48 hpf (Kajimura et al., 2005 (link); Kamei et al., 2008 (link)).

Astrocyte cultures were grown as described above. For OGD, cultures were exposed to 95 % nitrogen and 5 % CO₂ atmosphere maintained by constant gas flow at 37 °C in all experiments. Oxygen tension was kept at 5 % during the duration of the experiment. The Nitrogen/CO2 content was controlled with the ProOx P110 Oxygen Controller with an E702 Oxygen Sensor (model #P-110-E70) with single set point controller (#P110) (Biospherix). The media used in these OGD experiments consisted of hypoglycemic medium containing normal salts, 1 mg/mL bovine serum albumin (BSA) and 55 μM glucose (1 % of the normal glucose concentration). Control groups were grown under normal culture conditions (5 % CO₂ and 95 % Oxygen).