High-Resolution Respirometry in a Small-Volume Chamber

In this study, we want to investigate if the small volume chamber calibrated to 0.5 milliliter can be used in respirometric assays without compromising the accuracy obtained for the two milliliter classic volume in the Oroboros platform.

Our development of the small volume chamber and evaluation by high-resolution respirometry with rigorous quality control using precision OXPHOS analysis paved the way towards developments, new innovations, that is, number one, a 12-chamber O2k respirometer, number two, a robot for automatic titrations of substances in complex protocols applied for OXPHOS analysis. These applications will be crucial in further expanding towards mitochondrial analytics, diagnosis of mitochondrial diseases, evaluation of drug developments focusing both on the benefits of the drugs for mitochondrial function and on off-target effects that disrupt mitochondrial function involuntarily. When my team developed high-resolution respirometry more than 30 years ago, there was a paradigm that mitochondria, mitochondria, mitochondria. Maybe there was a difference between muscle and liver mitochondria, but that was it. Nobody was really interested in mitochondria outside of these paradigms. With our protocols developed for high-resolution respirometry, we showed a huge diversity of mitochondrial function. That allows us to evaluate mitochondrial respiratory control in diverse cell types, tissues, and species with a new eye. For 30 years ago, there were about 10 publications appreciating this. Now, we have with our instrument more than 1,500 using these protocols.

Four times less sample is required in the small volume chamber compared to the classic two milliliter chamber. In case of sample with low respiratory rates, the same amount of sample can be used to increase the volume specific oxygen fluxes to reduce the uncertainty of the measurement.

The focus of our work in the Oroboros R&D Laboratory is to combine instrumental development and academic research. We evaluate mitochondrial respiratory function and biogenetics profilings with multi-sensor analysis. That is measuring respiration, redox potentials, membrane potential, calcium uptake, hydrogen peroxide production, and various other parameters that allow us to have a deeper insight into the biogenetic profile and mitochondrial function and dysfunction.

[Narrator] To begin, mount the Small Volume, or SV chamber, onto the Oroboros. Then switch on the Oroboros, and click Connect to connect the Oroboros to the DatLab software. After drying the chambers completely, use a pipette to add 0.54 milliliters of water into the SV chamber. Then switch on the stirrers. Before closing the chamber, moisten the O-rings of the stoppers, keeping the capillary dry. Loosen the volume calibration ring, then insert the stopper downward until liquid fills the stopper capillary and a meniscus forms in the receptacle at the upper capillary end. Tighten the volume calibration ring to secure its position, ensuring calibrated volume consistency for consecutive insertions. To begin, fill the chambers with the respiration medium MiR05 Start the DatLab software. Select the appropriate protocol for the instrumental background test. Set the stirring rotation speed to 550 RPM, the chamber volume to 0.5 milliliters, the data recording interval at two seconds, and the experimental temperature to 37 degrees Celsius. Run the stirrer test and the air calibration. Set the R1 mark on the oxygen concentration plot by moving the cursor while holding the key Shift. Occasionally, conduct zero oxygen calibrations. During air calibration, prepare a fresh sodium dithionite stock solution by dissolving the O2-Zero Powder in 50 millimolar phosphate buffer to a concentration of 2.5 millimolar for the SV. After completing air calibration, close the chamber. Monitor the oxygen signal for approximately 60 to 120 minutes, allowing the oxygen concentration to decrease from air saturation to around 150 micromolar. Set four marks labeled J01 to J04 on the oxygen slope negative plot. Optionally, using a Hamilton syringe, titrate the sodium dithionite solution to adjust the oxygen concentration to 100 micromolar and set the mark J05. To calculate the instrumental oxygen background slope in DatLab, select Flux Slope. Next, open the oxygen background correction window and select Active file as the source of the oxygen background data. Check that all the J0 marks are used for the calculation of the linear regression. Then click Apply for the oxygen background correction. To begin, wash the Oroboros chambers with deionized water, and add fresh MiR05 medium into the chamber. After the instrumental background test, open a new DatLab tab and select the SUIT-003 protocol for human platelets experiments. Using the DatLab protocol overview that appears in the Protocol sidebar, select the chemical stock solutions. Then select the Hamilton syringes according to the chemicals. Place the syringes on the syringe rack with labels in sequence according to the SUIT protocol. For partial volume replacement, calculate the volume V in equivalent to the volume V out removed from the actual volume in the open chamber, factoring in the capillary dead volume. Then remove the fully inserted stopper and place it on the rack. Remove the calculated volume V out of MiR05 from the chamber based on the cell type and concentration of the stock cell suspension. Now, pipette an equivalent volume of the stock cell suspension with a known cell concentration into the chamber. In contrast, for complete volume replacement, siphon off the entire medium from the chamber. Add 0.54 milliliters of the stock of isolated cardiac mitochondria to the SV chamber. Close the chamber and siphon off any excess sample solution from the stopper receptacle. To start a Substrate Uncoupler Inhibitor Titration, or SUIT, protocol, open the Event window in DatLab to visualize the titration volumes. Using the Hamilton micro-syringe, titrate the chemical stock solutions into the chamber. Then set the event in DatLab. Allow for the flux stabilization after the titration. For data analysis, set a mark on the representative and stable portion of the plot. The background oxygen flux at air saturation was 3.9 times higher in the 0.5 milliliter chamber compared to the 2.0 milliliter chamber, consistent with the theoretically expected four-fold difference in volume specific background oxygen flux. The variability of instrumental background fluxes was nearly identical in the two chamber volumes. Respiratory rates of living platelets, permeabilized fibroblasts, and isolated mitochondria were closely correlated in both chamber types when the appropriate background correction was applied. Oxygen flux stabilized faster in the two milliliter chamber than in the 0.5 milliliter chamber post-reoxygenation. Residual oxygen consumption post-Complex III inhibition was consistent between the two chambers, aligning with the instrumental detection limit.