Continuous measurement of oxygen consumption rate in hypoxic conditions with adherent cells using an oxygen-sensitive optode

Cellular oxygen consumption rate (OCR) is a widely used readout of mitochondrial respiration and oxidative metabolism. However, many OCR assay workflows are limited by short assay durations, expensive specialized instrumentation, and reduced flexibility under controlled low-oxygen conditions. Here, we describe an in-incubator method for continuous, non-disruptive OCR measurement in adherent cell cultures using the PreSens OxoDish optical oxygen-sensing system. OCR is estimated by applying the steady-state solution of Fick's law to pericellular oxygen measurements acquired at well bottoms. This approach enables OCR monitoring over several hours to 24 hours without removing plates from a standard cell-culture incubator. Using MCF-7 breast cancer cells cultured in DMEM, we calculated basal OCR values of 20.07-32.79 amol cell-1s-1, similar to published values obtained using other methods. We also demonstrate a respiration profiling approach analogous to SeahorseXF Mito Stress Tests by quantifying OCR in parallel wells exposed to oligomycin, FCCP, or antimycin A/rotenone. Importantly, we demonstrate that the method remains functional under hypoxia (1.5% O2) without modification. Benchmarking the PreSens system against the SeahorseXF yielded values within the expected reported range. The approach we describe provides an accessible option for continuous OCR measurements in adherent cell cultures under a range of oxygen levels.