메나디온에 의한 혈소판 내 칼슘 변화측정시 형광 색소 사용의 문제점

Infeasibility of Measuring Ca2+ in Menadione-Exposed Platelets Using Fluorescent Dyes

It has been reported that dose-dependent Ca2+ increase by menadione in platelets could be measured by fluorescent dye, quin-2. The problems will be described here relating to measuring Ca2+ in menadione-exposed platelets using fura-2 and fluo-3, widely used fluorescent indicators. Additions of menadione to fura-2 loaded platelets and their lysates resulted in marked reduction in fluorescence intensity at both 340nm (Ca2+-unbound form) excitation wavelengths. Fura-2 excitation spectra were overlapped with UV-visible absorption spectra of menadione, suggesting that light absorption by menadione itself could quench fluorescence generated by fura-2. Next approach was to use fluo-3 which has the higher wavelength (490nm) of excitation. Previous work demonstrated that treatment with probenecid to platelets was required to prevent fluo-3 dye leakage. However, probenecid itself was proven to be inadequate to measure the concentration of intracellular Ca2+ by reducing menadione-induced cytotoxicity in platelets. Our results suggest that it is not feasible to measure Ca2+ in platelets by using fura-2 and fluo-3 in the presence of probenecid, and cautions should be taken to measure changes of intracellular Ca2+ levels by fluorescent dyes following chemical exposure.