Applications: Flow, ICC/IF, Func
Description
Hypoxia is a physiological state characterized by low oxygen levels in tissues and organs. Decreased oxygen levels can affect biological and cellular processes such as cell growth, proliferation, differentiation, metabolism, and angiogenesis (1). Therefore, hypoxia is also a common feature of many malignancies such as various cancers and inflammatory diseases (1,2). Hypoxia is regulated by hypoxia-inducible factors (HIFs). Under normoxia, HIFs are targeted for ubiquitin degradation initiated by prolyl hydroxylases (PHDs); however, in hypoxic conditions PHDs are inhibited and the stabilized HIFs can translocate to the nucleus and bind to hypoxia-responsive elements (HREs) (1,2). Methods to detect and monitor hypoxia include using antibodies to target HIF proteins or other proteins regulated by HIFs, as well as fluorescent oxygen-sensitive dyes and probes.
HypoxiTRAK(TM) is a cell-permeable, far-red fluorescent dye molecule that is used to assess hypoxia status in live cells. It can be used with both flow cytometry and immunocytochemical (ICC)/immunofluorescence (IF) applications. It shares spectral characteristics with DRAQ5(TM) and is compatible with conventional visible range fluorophores such as FITC and R-PE. Under lower oxygen conditions, HypoxiTRAK(TM) is converted into a metabolite expressing fluorescence as a hypoxia indicator. Mechanistically, it stops cell cycle progression, allowing for quantitative analysis of the number of hypoxic cells. If cells are sensitive cell-cycle arrest, HypoxiTRAK(TM) may cause cell death. A benefit of HypoxiTRAK(TM) is that it evaluates hypoxia without the need of primary antibodies or cell processing. The dye metabolite persists for several days and has a maximum excitation wavelength of 647 nm and emission wavelength at 697 nm. Additionally, it is non-toxic to cells in normoxic conditions. A practical application using of HypoxiTRAK(TM) in research is shown in a study where it was used to detect hypoxic regions in live head and neck squamous cell carcinoma (HNSCC) multicellular tumor spheroid (MCTS) cultures over time (3).
References
1. Tirpe AA, Gulei D, Ciortea SM, Crivii C, Berindan-Neagoe I. Hypoxia: Overview on Hypoxia-Mediated Mechanisms with a Focus on the Role of HIF Genes. Int J Mol Sci. 2019;20(24):6140. Published 2019 Dec 5. https://doi.org/10.3390/ijms20246140
2. Eltzschig HK, Carmeliet P. Hypoxia and inflammation. N Engl J Med. 2011;364(7):656-665. https://doi.org/10.1056/NEJMra0910283
3. Close DA, Johnston PA. Detection and impact of hypoxic regions in multicellular tumor spheroid cultures formed by head and neck squamous cell carcinoma cells lines. SLAS Discov. 2022;27(1):39-54. https://doi.org/10.1016/j.slasd.2021.10.008
