Ratio Fluorescence System Detects [ca ~ (2 +)] _i Factors Affecting Analysis And Application

Calcium was an important intracellular second messenger which played a key role in numerous cellular processes, such as cell proliferation, division, apoptosis and so on. Various methods and techniques for measurement of intracellular free calcium ([Ca²⁺]_i) were developed for recent years. Ratiometric fluorescence technique was one of the popular methods for [Ca²⁺]_i measurement.In this paper, various methods and techniques were introduced first; the advantages and the improvement of the ratiometric fluorescence system were enumerated, thus the purpose and significance for this paper were presented.In this paper, the mostly researches as follow:1. The parameters of loading time and loading concentration for fluorescence probe (Fura-2/AM) were screened in Hepa1-6 cells. The best loading parameters, 5μmol/L and 30min, were determined by experiments.2. R_max and R_min, the parameters in formula for [Ca²⁺]_i, were calculated in vivo. And the factors which were likely to influence the parameters were analyzed, which made calibration in measurement more precise. The results were more close to reality ofcells. Finally, R_max and R_min were calculated separately by analysis on the results of different cell density and loading concentration.3. The effect of KCl on non-excitable cells was analyzed by improved system. The results showed that 10mmol/L of KCl could make [Ca²⁺]_i of Hepa1-6 cells rise. And compared with 10mmol/L of KCl, there were no significant differences of the change of [Ca²⁺]_i aroused by 30mmol/L and 50mmol/L of KCl.