JSON
Rawle RJ, Villamil Giraldo AM, Boxer SG, Kasson PM
Biophys. J. 117 (3) 445-452 [2019-08-06; online 2019-07-02]
Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, whereas Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.
PubMed 31326109
DOI 10.1016/j.bpj.2019.06.022
Crossref 10.1016/j.bpj.2019.06.022
pmc: PMC6697364
pii: S0006-3495(19)30511-9