Singlet oxygen detection in vivo is hindered by nonspecific SOSG staining

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 ¹O₂. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo ¹O₂ detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of ¹O₂ 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 ¹O₂. 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.