Fluorescent hypoxia probe for flow cytometry

Hypoxia is a condition where low levels of oxygen levels are present (1% to 2% O 2 ). Hypoxia play a wide role in physiological and pathological conditions, from developmental angiogenesis to tumor progression and evasion. Hypoxia also modulates a number of immune functions from promoting inflammation to suppressing adaptive immunity. The current method for detecting hypoxic cells relies on an immunochemical approach. Pimonidazole react with peptide thiols in hypoxic cells and the resulting adduct is detected with an anti-pimonidazole antibody. To increase the availability of hypoxia detection reagents, we have developed a rhodamine-based hypoxia reagent (HR) for live cells. Using Jurkat leukemia cells incubated in hypoxic or normoxic conditions and stained with HR, we were able to clearly distinguish hypoxic cells from normoxic cells on a flow cytometer. We were also able to resolve cells incubated at differing levels of O 2 (10%, 5% and 2.5% O 2 ). To demonstrate that HR is compatible with other reagents, hypoxic cells were co-stained with a viability dye (SYTOX Red), a dye retained in intact mitochondria (TMRM) and with staining for a marker of apoptosis (annexin V). The results show that hypoxic cells excluded the dead cells dye while retaining TMRM, and were negative for annexin V staining at early time points. As expected, prolong incubation at hypoxia resulted in dead and apoptotic cells as evident in an increase in dead cells staining, a loss of mitochondrial integrity and an increase in annexin V + cells. In summary, we present a sensitive reagent for detecting hypoxic cells without the need for cellular fixation and permeablization, while retaining it usage with other live cell flow cytometry detection reagents.