Platkov Max, Tirosh Reuven, Kaufman Menahem, Zurgil Naomi, Deutsch Mordechai
The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan 52900, Israel.
The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan 52900, Israel.
J Photochem Photobiol B. 2014 Nov;140:306-14. doi: 10.1016/j.jphotobiol.2014.08.016. Epub 2014 Sep 2.
ROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis.
Photobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H₂O₂). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant.
Fluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H₂O₂ at 50 μM. At higher H₂O₂ concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H₂O₂ in phosphate buffer saline (PBS), where the addition of H₂O₂ decreases the decay time, regardless of the irradiation dose used.
The anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods.
Assessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.
活性氧（ROS）参与多种生理和病理过程的调节。细胞凋亡和坏死是由活性氧浓度变化诱导的过程。本研究旨在检测和量化细胞在细胞凋亡和坏死范围内对ROS浓度变化的反应。
使用荧光底物荧光素的光漂白作为探针，检测单个Jurkat - T淋巴细胞和前列腺癌 - 3（PC - 3）细胞对过氧化氢（H₂O₂）诱导的氧化应激的反应。提出了一个动力学模型来描述由于光漂白、染料水解、流入和泄漏导致的细胞内染料量的变化，产生一个单一的时间依赖性衰减指数 + 常数。
荧光素光漂白受到控制并用于检测细胞内ROS。在50 μM H₂O₂孵育的细胞中，测量到细胞内荧光素荧光衰减时间增加（光漂白缓慢）。与在磷酸盐缓冲盐水（PBS）中荧光素和H₂O₂的体外结果相反，在更高的H₂O₂浓度下测量到衰减时间减少（光漂白快速），在体外实验中，无论使用何种照射剂量，添加H₂O₂都会降低衰减时间。
与溶液不同，细胞中光漂白速率的异常、ROS浓度依赖性降低可能表明细胞内氧化应激保护机制的活性受到调节，正如之前用其他方法所观察到的那样。
通过对ROS敏感荧光团荧光强度的时间衰减来评估光漂白，可能适用于监测细胞中的氧化应激或ROS相关过程。
