Cheng Hang, Munro Daniel, Pamenter Matthew E
Department of Biology, University of Ottawa, Ottawa, ON, Canada.
University of Ottawa Brain and Mind Research Institute, Ottawa, ON, Canada.
MethodsX. 2021 Sep 20;8:101520. doi: 10.1016/j.mex.2021.101520. eCollection 2021.
Mitochondria generate aerobic cellular energy (i.e., ATP) through the reduction of oxygen to water via oxidative phosphorylation. The efficiency of this pathway can be measured by the phosphate/oxygen (ATP/O) ratio, which is the amount of ATP produced per oxygen atom reduced. This ratio thus provides a measure of the efficiency of mitochondrial respiration that can be readily compared between species. The magnesium green (MgGr) fluorometric method permits easy measurement of ATP/O ratios from isolated mitochondria but the standard analysis approach employs an endpoint method to calculate ATP/O ratios. Here, we present a modified method of ATP/O calculation that permits dynamic observation of fluorescent measurements of the consumption of O (JO) and the production of ATP (JATP). Specifically, by substituting the slope of a straight line within a given period of time (seconds to minutes) with the slope of a tangent to each time point (per second), it is possible to evaluate JO, JATP and the ATP/O ratio in a dynamic manner.•Provides second-by-second visualization of ATP/O ratios throughout experiments vs. a single measurement.•Dynamic visualization allows for easy identification of outlying data and more accurate calculation of mean ATP/O ratios.
线粒体通过氧化磷酸化将氧气还原为水来产生有氧细胞能量（即三磷酸腺苷，ATP）。该途径的效率可以通过磷/氧（ATP/O）比值来衡量，即每还原一个氧原子所产生的ATP量。因此，这个比值提供了一种线粒体呼吸效率的衡量方法，不同物种之间可以很容易地进行比较。镁绿（MgGr）荧光法可以轻松测量分离线粒体的ATP/O比值，但标准分析方法采用终点法来计算ATP/O比值。在这里，我们提出了一种改进的ATP/O计算方法，该方法允许动态观察氧气消耗（JO）和ATP产生（JATP）的荧光测量。具体来说，通过用每个时间点（每秒）的切线斜率代替给定时间段（秒到分钟）内的直线斜率，可以动态评估JO、JATP和ATP/O比值。
• 在整个实验过程中提供ATP/O比值的逐秒可视化，而不是单次测量。
• 动态可视化便于识别异常数据，并更准确地计算平均ATP/O比值。
