Cited by 207Cancer, Hypoxia, and MetabolismOptical Imaging and Spectroscopy TechniquesAnalytical Chemistry and Sensors

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[1] Measurement of absolute oxygen levels in cells and tissues using oxygen sensors and 2-nitroimidazole EF5

Koch, Cameron J.·2002·Methods in Enzymology

DOI 10.1016/s0076-6879(02)52003-6

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Abstract

We have established basic methods, using quantitative measures of EF5 binding, to estimate the actual pO2 of cells and tissues. In situations where the tissue can be dissociated into single cells, or for cell cultures, we can measure the distribution of cellular binding rates using flow cytometry and these can be compared with cells treated under pO2S controlled by the spinner vial or thin-film methods in vitro. The flow cytometer is calibrated by staining V79 cells treated with EF5 under "standard" conditions. For intact tissues treated with EF5 in vivo, we need to correct for possible variations in drug exposure (AUC). Frozen sections are stained for EF5 binding and are analyzed by a sensitive (cooled) CCD camera with linear output vs fluorescence [figure: see text] input. The camera has very consistent sensitivity, but the entire optical system, including the camera, can be calibrated by an absolute fluorescence standard (dye in hemocytometer). This system can also be used to measure the fluorescence of the flow cytometer standards, providing a direct link between the two assays. We can measure the maximum binding rate using the tissue cube method, but need to assume an "average" oxygen dependence of binding for intact tissues. The best-fit approximation for existing data is an inverse relationship between binding and pO2, with binding decreasing 50-fold between 0.1 and 10% oxygen. Using these methods, we routinely estimate the minimum pO2 (maximum binding) in experimental rodent and human tumors. In normal tissue models, an excellent correlation is found between near-maximal binding (severe hypoxia) and apoptosis (heart infarct and ductus arteriosus). Some normal tissues (e.g., skeletal muscle) are refractory to both cellular disaggregation and cube calibration methods. To extend the tissue imaging measurements to a complete two- or three-dimensional analysis of the distribution of tissue pO2s requires a substantial additional investment of imaging methods, which are currently being implemented.

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Authors: Koch, C. J.
Affiliations: University of Pennsylvania (US)
Corresponding author: Koch, Cameron J.
Venue: Methods in Enzymology
Publisher: Elsevier
Published: 2002
Volume: 352
Pages: 3-31
ISSN: 0076-6879 (ISSN-L)
DOI: 10.1016/s0076-6879(02)52003-6
OpenAlex: W1554643012
Cited by: 207
References: 78
Citations by year: 2026: 4 · 2025: 5 · 2024: 3 · 2023: 8 · 2022: 6 · 2021: 5 · 2020: 7 · 2019: 8 · 2018: 8 · 2017: 4 · 2016: 13 · 2015: 10 · 2014: 10 · 2013: 6 · 2012: 7
FWCI: 25.02
Type: book chapter
Language: en
Open access: closed
Locations: Methods in enzymology on CD-ROM/Methods in enzymology (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Optical Imaging and Spectroscopy Techniques
Physical Sciences › Chemistry › Analytical Chemistry › Analytical Chemistry and Sensors
Keywords: EF5, 2-nitroimidazole, tissue oxygenation, oxygen sensors, hypoxia, apoptosis, flow cytometry, tissue dissociation, fluorescence imaging, tumor oxygenation, V79 cells, ductus arteriosus, skeletal muscle
MeSH terms: Animals, Hypoxia, Biochemistry, Flow Cytometry, Fluorocarbons, Humans, Hydrocarbons, Fluorinated, Immunohistochemistry, Neoplasms, Oxygen, Pressure, Time Factors, Etanidazole, Mice

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[1] Measurement of absolute oxygen levels in cells and tissues using oxygen sensors and 2-nitroimidazole EF5

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