Quantitative Microplate-Based Respirometry with Correction for Oxygen Diffusion

Abstract

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O2 is consumed by the specimen, atmospheric O2 leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O2 but also store substantial amounts of gas. O2 flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O2] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O2]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

Ethanol Suppresses Ureagenesis in Rat Hepatocytes

“…The parameters of the measurement cycle were adjusted to avoid anoxia and were set as followings: Mix (4 min, during which the sensor plate moved up and down to equilibrate oxygen), Wait (30 s, during which oxygen sensors descended into wells to form 7-l measurement chambers above the plated hepatocytes), and Measure (1 min, 10 measurements of oxygen concentration to calculate respiratory rates) (35). Respiratory rates were calculated using Seahorse XF-24 software and the Direct ACOS fast algorithm with continuous averaging, as described (35). All incubations and measurements were done at 37°C.…”

Cytosolic Branched Chain Aminotransferase (BCATc) Regulates mTORC1 Signaling and Glycolytic Metabolism in CD4+ T Cells

“…8, A-D). ECAR provides a means to measure lactate production indirectly by measuring changes in pH associated with lactate release into the media (33,40). Non-glycolytic acidification was low and similar in WT and BCATc Ϫ/Ϫ cells (Fig.…”

Metabolic profile analysis of a single developing zebrafish embryo via monitoring of oxygen consumption rates within a microfluidic device

“…The measured [O 2 ] time-lapse data were filtered using a 7-point wide, second-order polynomial, first-order derivative Savitzky-Golay kernel (0.107, 0.071, 0.036, 0, À0.036, À0.071, À0.107). 13 This method provides additional smoothing of the calculated differentiated signal to alleviate the amplification of data noise during the -d[O 2 ]/dt calculation. Figure 5(b) shows the oxygen consumption rate of a single embryo over time (OCR(t), in pmol O 2 /min/embryo) at 3 hpf and 24 hpf inside a microwell for the 3 successive measurements that were performed by periodically sealing the glass microwell.…”