Optimization of Mitochondrial and Cytosolic pH Determination in Madin Darby Kidney Cells using CLSM Images

Reactive Oxygen Species (ROS) production and impairment of mitochondrial functioning is a central research topic. A recent review for Multiple Sclerosis1. Investigation of mitochondrial functioning in living cells under normal physiological conditions and metabolic inhibition provides biologically relevant information using confocal cell imaging2,3 Cytosolic calcium clearance does not necessarily lead to mitochondrial dysfunction as long as the mitochondrial matrix pH stayed acidified compared to the cytosol.2 Studying intra-mitochondrial versus cytosolic electrolyte homeostasis in living cells under metabolic stress might be crucial to understand the conditions where mitochondria become detrimental by producing ROS. Caveats are presented to properly obtain information about mitochondrial and cellular pH, structure and properties of the mitochondrial network under normal conditions and metabolic inhibition, by using optimized protocols4. The method critically depends on availability and selection of nuclear areas with lowest background fluorescence contribution while keeping experiment conditions rigorously similar. We present the influence of factors such as cell type, plating number, age of the cells, attachment surface properties, cell confluency, loading protocol of the dyes, background area selection on the values obtained. Optimization of parameters such as objective choice, detector voltage stability, fluorescence intensity saturation, illumination power and bleaching, imaging depth, point spread function (PSF) and reduction of cell motility are elucidated. Confluent Madin Darby Canine Kidney (MDCK) cells were loaded with mitotracker green and pH indicator SNARF. Images were collected with a Zeiss LSM 510 Meta CLSM. Data were analyzed with ImageJ4, and Matlab.1. Lassmann, H., and van Horssen, J., FEBS Lett. 20112. Smets, I. et al., Am. J. Physiol. Renal Physiol., 20043. Baron, Sz. et al., JASN, 20054. vandeVen, M, & Balut, C. et al. Methods Mol Biol. 2010