Oxygen supply of tissues is critical for their survival. Accordingly, fluctuations in oxygen partial pressure are linked to pathologic conditions such as renal failure, myocardial infarction as well as tumour progression. Fluctuating oxygen harms cells and tissue in two ways: during hypoxia in the course of blocked vessels, the lack of nutrients and oxygen (ischemia) impedes cells in generating energy. If this is followed by a rapid re-oxygenation (reperfusion), cells are exposed to increased oxidative stress, which induces inflammation and DNA damage. Despite being responsible for diseases, the biological effects of ischemia-reperfusion are hardly studied because of a lack of appropriate experimental models. A novel in vitro approach uses a microplate reader with atmospheric control unit (ACU) to measure cellular responses to ischemia-reperfusion conditions in high throughput and real time.
How does the Clariostar microplate reader mimic ischemia-reperfusion?
The microplate reader is able to rapidly return to physiological gas conditions upon active modification of O2 and CO2. The ACU regulates the CO2 and O2 concentration inside the reader. CO2 is required to stabilise the pH in most cell culture applications. It is directly pumped into the measurement chamber until the desired concentration is reached. Oxygen regulation is required to mimic its concentration changes during ischemia-reperfusion. As physiological oxygen concentrations are below ambient and because of oxygen’s contribution to explosions, molecular oxygen cannot be used to regulate oxygen inside the reader. Instead, nitrogen is used to displace O2 and to reduce the experimental O2 concentration. Nitrogen flooding allows a reduction from ambient to 0.2 % O2 with stable CO2 concentration in approximately 30 minutes. The oxygen reduction imitates the ischemic oxygen deprivation.
To quickly re-oxygenate the measurement chamber, ambient air is actively vented into the reader. In approximately 24 minutes the experimental O2 concentration rises from 0.2 % to 18 %. The rapid increase
is used to study reperfusion effects.
The Clariostar and its ACU were engineered to offer the highest flexibility. The ACU constantly measures CO2 and O2 concentrations in the reader and independently regulates both gases according to the target specified by the researcher. The experimental parameters O2 and CO2 concentration, decrease or increase of each gas as well as speed of gas changes can be programmed in the software of the microplate reader and are carried out automatically without any need for hands-on intervention.
Having these gas changes at the place of microplate measurement enables users to monitor cellular real-time responses employing microplate-compatible assays. A multimode plate reader is flexible to use and can combine any detection mode including fluorescence intensity, luminescence, absorbance, time-resolved fluorescence and more. Additional flexibility is provided by monochromator-based devices that are adjustable in the excitation and emission wavelengths in fluorescence measurements to support detection of varying fluorophores. The monochromator providing highest sensitivity in fluorescence intensity measurements is based on linear variable filters (LVF) that are slided one against the other to manage wavelength and bandwidth selection. The system exclusively used by the Clariostar increases sensitivity by higher light transmission and reduced stray light.
The novel platform for ischemia-reperfusion studies consisting of the Clariostar with ACU was used to determine the effect of rapid de- and re-oxygenation on cardiomyocytes. These cells are directly affected by myocardial infarcts and hence a major object of ischemia-reperfusion research.
To imitate ischemia/reperfusion, the ACU reduced the oxygen from ambient down to 1% and 50 min later back to ambient again (see grey line in Fig.1.). An intracellular phosphorescent probe that is sensitive to oxygen was used to report on intracellular oxygenation in iPSC-derived cardiomyocytes. The measurement combined MitoXpress-Intra (intracellular oxygenation), JC-1 (mitochondrial membrane potential) and DHE (reactive oxygen species). Cell oxygenation traces describe depth and duration of Cor.4U ischemia-reperfusion (A) with parallel monitoring of MMP and ROS (B). Respiring cells (untreated) showed reduced intracellular oxygen compared to the surrounding oxygen concentration because of oxygen consumption (Fig. 1, green). Upon ischemia, cells were completely deprived from oxygen until reperfusion. The homeostatic oxygenation was restored as the available oxygen had risen to 18 %. In non-respiring cells (Antimycin-treated), cellular oxygenation resembled the oxygen concentration regulated by the ACU (Fig. 1, purple). In the same experiment, the mitochondrial membrane potential as an indicator of oxidative phosphorylation and cell health was measured with the fluorescent dye JC1. This revealed MMP dissipation in non-respiring cells (Fig. 1B). As a third cellular response generation of reactive oxygen species was acquired with the redox-sensitive fluorophore dihydroethidium (DHE). Non-respiring cells showed a higher induction of reactive oxygen species than respiring cells (Fig. 1B).
The novel platform for assaying rapid oxygen changes such as ischemia-reperfusion reads any assay in any detection mode.It also extends this to define custom O2 and CO2 changes and obtain biological information under physiological gas conditions and high throughput.
Andrea Krumm is application specialist at BMG Labtech.
www.bmglabtech.com
