测定细胞内钙离子浓度的方法

【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