The effect of Antimycin and FCCP treatment on the oxygen levels at a HEK293T cell monolayer. Antimycin is an ETC inhibitor while FCCP uncouples oxygen consumption from ATP production. Initial probe signal reflects the steady state oxygen level lower than ambient oxygen due to cellular oxygen consumption. Antimycin treatment inhibits this consumption resulting in oxygen diffusing into the monolayer returning it to ambient concentrations. FCCP treatment causes an increase in oxygen consumption resulting in in a rapid decrease in the concentration of oxygen at the cell monolayer until a new steady state is reached.
Monitoring changes in monolayer oxygenation at decreasing oxygen concentration. Oxygen concentration is stepped between 21% and 1%, with an atmospheric control unit. The Intracellular Oxygen Concentration Assay signal reflects the actual oxygen concentrations at the cell monolayer. Note that cellular respiration drives the monolayer to much lower oxygen concentrations than those imposed.Note: Intracellular Oxygen Concentration Assay is simply added ~16 hours prior to drug treatment. The probe is stable and reversible, allowing real-time monitoring of (multiple) drug effects under controlled oxygen conditions.
HCT116 cells were treated with the agonist, Ryaniodine. Results indicate that with the Intracellular Oxygen Concentration Assay, changes in intracellular oxygen levels (or ETC) can be detected by lifetime measurements. The agonist will cause an increase in ETC activity, therefore increasing lifetime of the reagent, which suggests a decrease in intracellular oxygen levels.
The effect of HepG2 cells treated with an electron transport chain inhibitor (Antimycin) and uncoupler (FCCP). Consistency of response & inhibition and uncoupling of ETC activity (A) and kinetic analysis of metabolic responses agonist treatment (B).
FLIM image of a 96 Well Plate well seeded with HepG2 loaded with Intracellular Oxygen Concentration Assay (ab197245).