Developing assays to quantify function in cells and tissues

Chronically developing dysfunction may be hallmarked by subtle deviations from the normal. Detection of such subtle functional disturbances requires precise quantitative assays that are applicable to disease models or primary human tissues. Major challenges have limited bioenergetic studies in intact cells in the past, including requiring large amounts of specimen, encountering diffusional problems, and the plasma membrane’s action as a barrier between mitochondria and the detection system. Today, micro-scale, absolute quantitative technologies are available to track subtle bioenergetic changes, several of them developed at the Buck Institute.

Cell respirometry has been revolutionized by the Seahorse Extracellular Flux Analysis (developed originally by Seahorse Bioscience, Billerica, MA). We developed the oxygen consumption rate (OCR) calculation algorithm of the instrument, fixing distortions in the readout due to oxygen diffusion into the small sample¹. To date, this technology is the gold standard in cellular bioenergetics and it has been used in over 7000 publications. More recently, we extended this technology to quantify ATP fluxes in glycolysis and oxidative phosphorylation². Today, we use these technologies in conjunction with image-based cell number normalization and mitochondrial membrane potential assay to understand basic mechanisms how aging and type 2 diabetes detunes cellular energy metabolism³. We also provide these technologies to users of Buck Morphology and Imaging (Microscopy) Core.

The mitochondrial membrane potential (ΔψM) is a commonly assayed bioenergetic variable, but typical fluorescence assays in intact cells are prone to data misinterpretation due to factors other than ΔψM contributing to the readout⁴. We have developed and currently applying technology for the absolute and unbiased determination of ΔψM in monolayer cells or 3D tissue explants^4-6. This technology provides millivolts calibrated values for both plasma membrane potential (ΔψP) and ΔψM in intact, cultured or immobilized isolated live cells. The key advantage of this technology compared to semi-quantitative techniques is that ΔψM is determined in an absolute scale independently of cell geometry or ΔψP, allowing comparison of specimens where these confounders of typical ΔψM assays differ⁴. Calculation of millivolt values from fluorescence time courses is enabled by the Membrane Potential Calibration Wizard module in Image Analyst MKII, developed by Dr Gerencser.

Dr. Gerencser is the founder of Image Analyst Software and developed Image Analyst MKII, a microscopy image analysis tool for analysis of time lapse and multi-dimensional image data. This GUI-based application serves as a platform for all microscopy-based assaying we perform. The pipeline- and batch-based image analysis enables both manual exploration and automated analysis of larger image data sets. Image analysis pipelines in Image Analyst MKII allow fast prototyping and optimization by power users and presenting the analysis pipelines with “biologically decoded” select analysis parameters for general audience. Image Analyst MKII performs fully automated analysis of microplate-based recordings for the unbiased absolute ΔψM assay, including pre-processing, image registration, AI-based cell segmentations, calculation of fluorescence time courses and their conversion into millivolt values⁴.

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References

1. Gerencser, A. A. et al. Quantitative microplate-based respirometry with correction for oxygen diffusion. Analytical chemistry 81, 6868-6878 (2009). https://doi.org:10.1021/ac900881z
2. Mookerjee, S. A., Gerencser, A. A., Nicholls, D. G. & Brand, M. D. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. Journal of Biological Chemistry 292, 7189-7207 (2017). https://doi.org:10.1074/jbc.M116.774471
3. Gerencser, A. A. Metabolic activation-driven mitochondrial hyperpolarization predicts insulin secretion in human pancreatic beta-cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1859, 817-828 (2018). https://doi.org:10.1016/j.bbabio.2018.06.006
4. Lerner, C. A. & Gerencser, A. A. Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells. Methods Mol Biol 2497, 11-61 (2022). https://doi.org:10.1007/978-1-0716-2309-1_2
5. Gerencser, A. A., Mookerjee, S. A., Jastroch, M. & Brand, M. D. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells. PloS one 11, e0159199 (2016). https://doi.org:10.1371/journal.pone.0159199
6. Gerencser, A. A. et al. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. The Journal of physiology 590, 2845-2871 (2012). https://doi.org:10.1113/jphysiol.2012.228387