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        测定细胞内钙离子浓度的方法

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        38503

        【求助】测定细胞内钙离子浓度都有哪些方法?

        参与者:junxia

        想测定某一蛋白对细胞内钙离子浓度的影响。请教各位大虾都有哪些测定方法?好像常用的是共聚焦?除此之外呢?

        参与者:gzgygy

        单个观察用confocal。

        群体准确性最高的不是confocal而是流式。

        参与者:jc_484190

        对,流式是不错的,建议你可以试试,

        参与者:junxia

        楼上两位能提供流氏的参考文献或资料么?

        参与者:sherrie660117

        FLOW 可以用Indo-1(需紫外激光)和FLUO3(488nm)激光.参考文献较大

        告诉我EMAIL,发给你.

        参与者:sherrie660117

        昨天上传了几篇文献,怎么没有呀? 我再试试.

        1.caj (23.21k)

        参与者:sherrie660117

        昨天上传了几篇文献,怎么没有呀? 我再试试.

        计算公式.kdh (270.98k)

        参与者:sherrie660117

        昨天上传了几篇文献,怎么没有呀? 我再试试.

        細胞內钙离子流动实验.doc (24.5k)

        参与者:honvey

        以下是我的实验方法。用了流式和荧光分光光度计检测。

        2.9.Measurement of intracellular Ca2+

        Intracellular Ca2+ levels were determined with the Ca2+-sensititive fluorochrome Fluo-3/acetoxymethyl ester (Fluo-3/AM)by a Becton Dickinson FACS Calibur flow cytometer or a PerkinElmer LS 55 fluorescence spectrophotometer (Wang and Xu,2005).Cells were incubated with 3 uM Fluo-3/AM at 37 ℃ for 30 min in the dark after the treatment with salidroside plus H 2 O 2 as described above.Cells were then gently rinsed three times with D-Hanks&;rsquo; solution and the fluorescence was analyzed by flow cytometry.Fluo-3/AM binds cytoplasmic free calcium and emits a green fluorescence (peak at 526 nm)detected using FL1.

        Kinetic study of intracellular Ca2+ was conducted as follows.SH-SY5Y cells (3×;;106/ml)were stained with 3 ;;;Fluo-3/AM dye at 37 ℃ for 30 min,and then washed to remove extracellular Fluo-3/AM dye and resuspended in Krebs-Ringer-HEPES (KRH)buffer (131 mM NaCl,5 mM KCl,1.3 mM MgSO 4 ,1.3 mM CaCl 2 ,0.4 mM KH2PO 4 ,6 mM glucose,20 mM HEPES,pH 7.4).Cells were then treated with 150 ;M H 2 O 2 in the absence or presence of 10 or 100 ;M of salidroside for 6 minutes.Fluorescence was determined using a PerkinElmer LS 55 fluorescence spectrophotometer with excitation and emission wavelengths of 488nm and 526 nm every 1 min.Intracellular Ca2+ was calculated from the fluo-3 fluoresce intensity using the equation: [Ca2+]i = Kd [(F - Fmin)/(Fmax - F)],where Kd = 400 nM (Okamoto et al.,1997).The maximal Fluo-3 fluorescence intensity (Fmax)was determined by adding 0.1% Triton X-100,and the minimal fluorescence (Fmin)was determined by quenching Fluo-3 fluorescence with the addition of 5 mM EGTA.F is the fluorescence measured without the addition of Triton-X-100 or EGTA.

        参与者:pxy999

        请问sherrue6601,honvey,若用FLUO-3测细胞内钙并想知道细胞内钙的浓度,怎办?看完文献后,发现用Triton-100和EGTA来测定细胞内钙的最大值和最小值!我的问题是这两种试剂什么时间与待测细胞混合?谢谢。

        FLuo-3的步骤:

        1。D&;rsquo;-HANKS清洗在三次,加2&;mu;m的Fluo-3/AM,避光,37度,30分钟

        2。细胞负载后,用D&;rsquo;-HANKS清洗二次

        3。在confocal上分析细胞内钙离子浓度

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