Detection of reactive oxygen species induced by radiation in cells using the dichlorofluorescein assay
Detection of reactive oxygen species induced by radiation in cells using the dichlorofluorescein assay
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DOI：
10.1667/rr0925.1
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发表时间：
2007-08-01
影响因子：
3.4
通讯作者：
Emel'yanov, Maksim O.
中科院分区：
文献类型：
--
作者：
Korystov, Yuri N.;Shaposhnikova, Vera V.;Emel'yanov, Maksim O.
本研究的目的是使用2 '7'-二氯荧光素（DCF）测定法测定在含有血清的培养基中照射的细胞内产生的活性氧（ROS）的量。在MF 44 Perkin Elmer荧光计上记录细胞和培养基中的DCF荧光，并且在Partec流式细胞仪上仅记录细胞中的荧光。用剂量率为1.12戈伊/min的X射线照射人喉癌HEp-2细胞和淋巴白血病P388细胞，研究了温度、pH值、血清浓度等因素对2 '7'-二氯荧光素（DCFH）氧化为DCF的影响，以减少二氯荧光素测定ROS的误差。通过DCF测定在细胞中登记的ROS的量被发现依赖于在照射期间培养基中的血清浓度。在存在10%血清的情况下，辐射对可检测到的ROS的量没有影响。辐射对细胞内活性氧形成的影响几乎完全取消，如果照射后立即删除媒体辐射曝光。在具有低血清含量的培养基中照射的细胞中ROS形成的增加主要是由于在培养基中产生的水的辐解产物并氧化位于细胞中的DCFH。(c)2007年，辐射研究协会。
The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells. (c) 2007 by Radiation Research Society.