A simplified protocol to induce hypoxia in a standard incubator: A focus on retinal cells.

Hypoxia chambers have traditionally been used to induce hypoxia in cell cultures. Cellular responses to hypoxia can also be mimicked with the use of chemicals such as cobalt chloride (CoCl), which stabilizes hypoxia-inducible factor alpha-subunit proteins. In studies of ocular cells using primary cells and cell lines, such as Müller glial cell (MGC) lines, photoreceptor cell lines, retinal pigment epithelial (RPE) cell lines and retinoblastoma cell lines oxygen levels employed in hypoxia chambers range typically between 0.2% and 5% oxygen. For chemical induction of hypoxic response in these cells, the CoCl concentrations used typically range from 100 to 600 μM. Here, we describe simplified protocols for stabilizing cellular hypoxia-inducible factor-1α (HIF-1α) in cell culture using either a hypoxia chamber or CoCl. In addition, we also provide a detailed methodology to confirm hypoxia induction by the assessment of protein levels of HIF-1α, which accumulates in response to hypoxic conditions. Furthermore, we provide a summary of conditions applied in previous studies of ocular cells.