【求助】測(cè)定細(xì)胞內(nèi)鈣離子濃度都有哪些方法？

參與者：junxia

想測(cè)定某一蛋白對(duì)細(xì)胞內(nèi)鈣離子濃度的影響。請(qǐng)教各位大蝦都有哪些測(cè)定方法？好像常用的是共聚焦？除此之外呢？

參與者：gzgygy

單個(gè)觀察用confocal。

群體準(zhǔn)確性最高的不是confocal而是流式。

參與者：jc_484190

對(duì)，流式是不錯(cuò)的，建議你可以試試，

參與者：junxia

樓上兩位能提供流氏的參考文獻(xiàn)或資料么？

參與者：sherrie660117

FLOW 可以用Indo-1（需紫外激光）和FLUO3（488nm）激光.參考文獻(xiàn)較大

告訴我EMAIL，發(fā)給你.

參與者：sherrie660117

昨天上傳了幾篇文獻(xiàn)，怎么沒有呀? 我再試試.

1.caj （23.21k）

參與者：sherrie660117

昨天上傳了幾篇文獻(xiàn)，怎么沒有呀? 我再試試.

計(jì)算公式.kdh （270.98k）

參與者：sherrie660117

昨天上傳了幾篇文獻(xiàn)，怎么沒有呀? 我再試試.

細(xì)胞內(nèi)鈣離子流動(dòng)實(shí)驗(yàn).doc （24.5k）

參與者：honvey

以下是我的實(shí)驗(yàn)方法。用了流式和熒光分光光度計(jì)檢測(cè)。

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.

參與者：pxy999

請(qǐng)問sherrue6601，honvey，若用FLUO-3測(cè)細(xì)胞內(nèi)鈣并想知道細(xì)胞內(nèi)鈣的濃度，怎辦？看完文獻(xiàn)后，發(fā)現(xiàn)用Triton-100和EGTA來測(cè)定細(xì)胞內(nèi)鈣的最大值和最小值！我的問題是這兩種試劑什么時(shí)間與待測(cè)細(xì)胞混合？謝謝。

FLuo-3的步驟：

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

2。細(xì)胞負(fù)載后，用D&；rsquo；-HANKS清洗二次

3。在confocal上分析細(xì)胞內(nèi)鈣離子濃度