Department of Biochemistry, Faculty of Medicine, Kuwait University, 13110, Kuwait, Kuwait.
Sci Rep. 2024 Sep 5;14(1):20669. doi: 10.1038/s41598-024-71801-9.
Singlet oxygen is considered an important cell damaging agent due to its propensity to react with organic compounds. This drives the interest in developing methods for determination of O. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo O detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of O involvement in cell and tissue damage. Our investigation, however, demonstrate that different microbial species and cancer cells become fluorescent when exposed to SOSG under conditions which exclude generation of O. Cells, permeabilized with chlorhexidine or by heat exposure under anaerobic conditions, exhibited SOSG fluorescence. Permeabilized cells could be stained with SOSG even 24 h post-permeabilization. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was plasma membrane damage. Spectral analyses of different batches of SOSG revealed that SOSG endoperoxide (SOSG-EP) did not increase even after prolonged storage under the recommended conditions. The commercial preparations of SOSG, however, were not SOSG-EP free, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen production in vivo.
单线态氧被认为是一种重要的细胞损伤剂，因为它容易与有机化合物发生反应。这激发了人们开发用于测定单线态氧的方法的兴趣。由于应用简单和高灵敏度，荧光探针成为体内 O 检测的热门选择。尽管商业上可用的单线态氧传感器绿（SOSG）被宣称具有细胞非渗透性，但它被广泛应用于支持 O 参与细胞和组织损伤的断言。然而，我们的研究表明，在排除单线态氧生成的条件下，不同的微生物物种和癌细胞在暴露于 SOSG 时会发出荧光。用洗必泰或在厌氧条件下热暴露使细胞通透化，会使细胞显示 SOSG 荧光。即使在通透化 24 小时后，通透化的细胞也可以用 SOSG 染色。由于 SOSG 是细胞非渗透性的，导致荧光染色的主要因素是质膜损伤。对不同批次的 SOSG 的光谱分析表明，即使在推荐条件下长时间储存，SOSG 内过氧化物（SOSG-EP）也不会增加。然而，商业上的 SOSG 制剂并不是 SOSG-EP 免费的，当 SOSG 染色被用作体内单线态氧产生的证明时，它会产生错误的结果。
