title: Analysis of a Nitroreductase-Based Hypoxia Sensor in Primary Neuronal Cultures.
abstract: The ability to assess oxygenation within living cells is much sought after to more deeply understand normal and pathological cell biology. Hypoxia Red manufactured by Enzo Life Sciences is advertised as a novel hypoxia detector dependent on nitroreducatase activity. We sought to use Hypoxia Red in primary neuronal cultures to test cell-to-cell metabolic variability in response to hypoxic stress. Neurons treated with 90 min of hypoxia were labeled with Hypoxia Red. We observed that, even under normoxic conditions neurons expressed fluorescence robustly. Analysis of the chemical reactions and biological underpinnings of this method revealed that the high uptake and reduction of the dye is due to active nitroreductases in normoxic cells that are independent of oxygen availability.
authors: Vanderbilt Neuroscience Graduate Program, ‡Departments of Chemistry, §Neurology, and ∥Pharmacology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: Britney N
authors: BN
authors: Lizama-Manibusan
authors: Vanderbilt Neuroscience Graduate Program, ‡Departments of Chemistry, §Neurology, and ∥Pharmacology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: Sharon
authors: S
authors: Klein
authors: Vanderbilt Neuroscience Graduate Program, ‡Departments of Chemistry, §Neurology, and ∥Pharmacology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: Jennifer R
authors: JR
authors: McKenzie
authors: Vanderbilt Neuroscience Graduate Program, ‡Departments of Chemistry, §Neurology, and ∥Pharmacology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: David E
authors: DE
authors: Cliffel
authors: Vanderbilt Neuroscience Graduate Program, ‡Departments of Chemistry, §Neurology, and ∥Pharmacology, Vanderbilt University , Nashville, Tennessee 37232, United States.
authors: BethAnn
authors: B
authors: McLaughlin
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000494:pharmacology; D002452:Cell Count; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005947:Glucose / Q000172:deficiency; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009601:Nitroreductases / Q000378:metabolism*; D010100:Oxygen; D016548:Prosencephalon / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005947:Glucose; D008869:Microtubule-Associated Proteins; D009601:Nitroreductases; D010100:Oxygen; D014404:Tubulin; D000099162:Endothelial PAS Domain-Containing Protein 1; C489787:MAP2 protein, rat
keywords: Fluorescence; hypoxia; nitroreductase; oxygen; oxygen and glucose deprivation