HypoxiTRAK™

Flow Cytometry: HypoxiTRAK(TM) [NBP3-18531] - Flow cytometry of -metabolite, blue & red excitation; intact A549 cells under hypoxia (3%, 1%), with & without [4 day co-exposure]

Immunocytochemistry/Immunofluorescence: HypoxiTRAK(TM) [NBP3-18531] - -metabolite accumulates in cytoplasm of A549 cells (ex. 633 nm; em. 680/20 nm) exposed to 100 nM for 4 days 1% O2

Flow Cytometry: HypoxiTRAK(TM) [NBP3-18531] - -metabolite persistence: intact A549 cells retain >70% after washing and 24h incubation in -free medium (ex. 633nm; em. >695nm)

HypoxiTRAK(TM) [NBP3-18531] - Intracellular accumulation of HypoxiTRAK(TM)-metabolite is sensitive to degree of hypoxia and [HypoxiTRAK] (A549 cells, conditions as image 2)

Immunocytochemistry/Immunofluorescence: HypoxiTRAK(TM) [NBP3-18531] - Multi-Cellular Tumor Spheroid (MCTS) generated from 5000 cells (Cal33, HNC) in a ULA well. MCTS grown for 3 days then probed with 50 nM HypoxiTRAK(TM) for a further 3 days. False-color heatmap image shows hypoxia reported in a cortical pattern and with a hypocellular centre (by Hoechst staining consistent with controls, not shown).Courtesy of DA Close & PA Johnston - University of Pittsburgh, School of Pharmacy.

• Applications: Flow, ICC/IF, Func

HypoxiTRAK™ Summary

• Description: HypoxiTRAK(TM) is a novel, far-red fluorescing dye that reveals hypoxic experience of individual cells yet is non-toxic to normoxic cells. It is suitable for flow cytometry and imaging protocols and is compatible with common vis. range fluors e.g. FITC, R-PE. HypoxiTRAK(TM) enables entirely new assay approaches to functional hypoxia.
  
  HypoxiTRAK(TM) can be used in flow cytometry and fluorescence imaging and is spectrally compatible with common visible range fluors including FITC and R-PE.
  
  HypoxiTRAK(TM) is supplied as a faint blue aqueous solution at a concentration of 10uM. Products are shipped at ambient temperature, but on receipt packs should be stored at 2-8C. DO NOT FREEZE! Otherwise HypoxiTRAK(TM) can come out of solution when frozen and it is difficult (but not impossible) to get it back into solution.
  HypoxiTRAK(TM) can be diluted in culture media (e.g. RPMI 1640) and physiological buffers (eg PBS, Hanks's, etc.).

Applications/Dilutions

• Dilutions:
  • Flow Cytometry
  • Functional Assay
  • Immunocytochemistry/ Immunofluorescence
• Application Notes: Flow Cytometry: suitable for dynamic live cell hypoxia study; extract statistical information on the distribution of hypoxic experience over both time and treatment conditions Immunocytochemistry/Immunofluorescence: dynamic live cell hypoxia study; immuno-fluorescence; gain understanding of the spatial hypoxic experience in micro-tissues and spheroids

Packaging, Storage & Formulations

• Storage: Store at 4C in the dark. Do not freeze.

Notes

HypoxiTRAK(TM) is a registered trademark of BioStatus Limited.

Background

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

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Support products are guaranteed for 6 months from date of receipt.