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 °C for 30 min in the dark after the treatment with salidroside plus H2O2 as described above. Cells were then gently rinsed three times with D-Hanks’ 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 °C 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 MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, 6 mM glucose, 20 mM HEPES, pH 7.4). Cells were then treated with 150 M H2O2 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.
最新回复
junhun (2012-3-10 16:56:47)
单个观察用confocal。
群体准确性最高的不是confocal而是流式。
8princess8 (2012-3-10 16:57:09)
对，流式是不错的，建议你可以试试。
nn255 (2012-3-10 16:57:51)
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 °C for 30 min in the dark after the treatment with salidroside plus H2O2 as described above. Cells were then gently rinsed three times with D-Hanks’ 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 °C 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 MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, 6 mM glucose, 20 mM HEPES, pH 7.4). Cells were then treated with 150 M H2O2 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.
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小糖块 (2012-3-10 16:58:17)
FLuo-3的步骤：
1。D’-HANKS清洗在三次，加2um的Fluo-3/AM,避光，37度，30分钟
2。细胞负载后，用D’-HANKS清洗二次
3。在confocal上分析细胞内钙离子浓度
c86v (2012-3-10 16:58:37)
[求助]测定细胞内钙离子浓度都有哪些方法？