Intracellular free calcium in Fura-2 Fluorescence.docVIP

 PAGE \* MERGEFORMAT 8 Intracellular free calcium in Fura-2 Fluorescence [Keywords:] rat cardiomyocytes Fura-2 in the chemical name 2 - [6 - Double acid base -5-- (2 - double amino acid) -5-- toluene oxygen ethoxy] -2-- benzo furyl -5-- oxazolidine carboxylic acid potassium Five salt. belong to the second generation of fluorescent indicator, is to determine the importance of intracellular calcium reagent [1]. with the Ca2 + fluorescent indicator Fura-2 detected in the cytoplasm of living cells [Ca2 +] i rise than a decade of a technology , has been widely used in cell physiology, pathology studies have been reported in the literature used to determine cell [2], tissue [3] and so the free calcium. application of the Court’s 970CRT fluorescence spectrophotometer, Based on our experiments conditions established in this study using Fura-2 dual-wavelength fluorescence spectrometry intracellular calcium method. 1 Materials and methods 1.1 Instruments and reagents 970CRT fluorescence spectrophotometer (Shanghai Analytical Instruments Factory, IGO150 type CO2 incubator (Thermo electron corporation, 601 super-heated water bath incubator (Jintan Medical Instrument Factory, TDZ5-WS small centrifuge (Beijing Optical Instrument Factory. Fura-2/AM, DMEM, 1:250 trypsin was purchased from SIGMA Corporation, TritonX-100, EDTA were purchased from Shanghai Huamei, calf serum was purchased from Beijing Institute of Biotechnology Hengsheng Ma Yuan, the other reagents were of analytical grade . 1.2 Experimental Methods 1.2.1 Principle Fura-2 has a strong hydrophilic, easily through the cell membrane, but with the Ca2 + specific binding to a specific wavelength of the fluorescence under UV excitation, acetyl carboxyl methyl ester (Acetoxy-Methylester, AM with the lipophilic, can enter the cell through the cell membrane. cytoplasm can be Fura-2/AM esterase hydrolysis of free Fura-2, Fura-2 and the intracellular free Ca2 + to form Fura-2-Ca2 + complexes , Fura-2 an