Petr M J, Wurster R D
Department of Neurological Surgery, Loyola University Medical Center, Maywood, Illinois 60153, USA.
Cell Calcium. 1997 Mar;21(3):233-40. doi: 10.1016/s0143-4160(97)90047-6.
Accurate estimates of cytosolic free Ca2+ with fluorescence indicators are dependent on the determination of the in situ dissociation constants (kd) of the intracellular dyes and the correction for background fluorescence. The in situ dissociation constant for Ca2+ and indicator dye Fura-2/AM varies significantly from the in vitro published values due to differences in ionic strength, pH, viscosity and Ca2+ buffering by intracellular lipids and proteins. During the course of a measurement, background fluorescence changes may occur as the result of endogenous fluorescent compounds and compartmentalized Fura-2 indicator. The in situ dissociation constant value was determined for human astrocyte cell line U373-MG by creating several known intracellular Ca2+ concentrations while measuring total fluorescence and background fluorescence values for each. The background fluorescence was not constant, rather it demonstrated a linear relationship with the free cytosolic Ca2+ concentrations and total fluorescence intensities. The Ca2+ dependent and total fluorescence dependent background was expressed as a linear equation and subtracted appropriately from the total intensity measurements. The in situ dissociation constant was determined to be 3-fold greater than in vitro measurements after the background was corrected. The experimentally determined standard linear equations for background quantitation and the in situ dissociation constant for this line produce accurate cytosolic free Ca2+ estimates.
使用荧光指示剂准确估计胞质游离钙离子（Ca2+），依赖于细胞内染料原位解离常数（kd）的测定以及背景荧光的校正。由于离子强度、pH值、粘度以及细胞内脂质和蛋白质对Ca2+的缓冲作用存在差异，Ca2+与指示剂染料Fura-2/AM的原位解离常数与体外公布的值有显著不同。在测量过程中，由于内源性荧光化合物和分隔的Fura-2指示剂，背景荧光可能会发生变化。通过创建几个已知的细胞内Ca2+浓度，同时测量每个浓度下的总荧光和背景荧光值，来确定人星形胶质细胞系U373-MG的原位解离常数。背景荧光并非恒定不变，而是与游离胞质Ca2+浓度和总荧光强度呈线性关系。将Ca2+依赖性和总荧光依赖性背景表示为线性方程，并从总强度测量值中适当减去。校正背景后，确定原位解离常数比体外测量值大3倍。通过实验确定的该细胞系背景定量标准线性方程和原位解离常数，可准确估计胞质游离Ca2+。
