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AAT Bioquest®钙离子检测探针,39.7分的文章,想发高分的看过来

作者:admin信息来源:达科为日期:2018年01月30日打印字体:  
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70年代初,在Ca2+受体蛋白-钙调素(CaM)的发现极其功能的研究基础上提出Ca2+第二信使学说。细胞内Ca2+的分布与转移是形成Ca2+信号的基础,因此监测Ca2+的动态变化是研究G蛋白偶联受体(GPCR)有效刺激剂的重要手段。


AAT Bioquest 提供各种优质钙离子检测探针

1   Fluo8

Fluo-3和Fluo-4是常用的可见光激发的Ca2+指示剂,然而Fluo-3AM和Fluo-4 在体内结合钙离子后的荧光强度不高,需要比较苛刻的loading condition来加强荧光强度。AAT Bioquest®Fluo-8改善了二者的loading condition和荧光强度问题,比如Fluo-8只需要在室温孵育,而Fluo-3和Fluo-4需要37度水浴,另外Fluo-8的荧光强度是Fluo-4的两倍,是Fluo-3的4倍,另外Fluo-8依旧保持Fluo-3和Fluo-4的通道。

Figure 2. U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells

were incubated with 100 µL of 4 µM Fluo-3 AM, Fluo-4 AM and Fluo-8® AM (Cat# 21080) in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged

with a fluorescence microscope using FITC channel.

 

2 Calbryte 系列



为什么Calbryte™系列能够提高信噪比


首先Calbryte™系列被偶联了乙酰氧基甲酯( acetoxymethyl ,AM),意义有二:一是利用AM的亲酯性使得探针能够顺利进入到细胞内;二是AM基团保证了Calbryte™ 荧光探针在没有进入细胞之前的非活性以及非荧光的特性,这样就能减少非特异性信号,提高信噪比。

 

     

为什么Calbryte™系列荧光强度更强?



因为该系列染料有更高的量子产率,是Fluo-3 or Fluo-4.的3倍,什么意思呢,就是吸收同样数目的光子,Calbryte™系列能发出的荧光强度是它们的3倍,而且激发光同样是常见的499nm。

QY = Fluorescence Quantum Yield in the presence of 5 mM calcium citrate



这里跟大家提一个名词,p-糖蛋白,p-glycoprotein 1 ,P-gp),在很多细胞里比如Hella,P-gp扮演着ATP依赖的离子泵将很多小分子泵出细胞外,当然进入细胞的钙离子探针也免不了这种遭遇。这就导致被排到细胞外的探针会结合胞外的离子,造成背景值增高而胞内的目的信号变弱,信噪比降低。有的会加入丙磺舒(probenecid),一种通道抑制剂来减少这种泄露。但是这种物质的添加并不理想,有报道TRPV2受体会被其激活而TAS2R 受体又会被其抑制,因此会给研究带来不可预知的可变因素。

而Calbryte™ dyes却能够通过平衡离子电量来摆脱窘境。进入细胞后一旦被水解,便形成带有大量负电荷的亲水性分子大大降低了这种泄露。

 

Carbachol dose response was measured in CHO-M1 cells with Calbryte™ 520 AM and Fluo-4 AM. CHO-M1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 520 AM in HH Buffer or 10 µg/ml Fluo-4 in HH Buffer was added and incubated for 45 minutes at 37 °C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. Carbachol (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.

 

另外,AAT Bioquest® 提供无probenecid、无需洗涤的钙离子检测试剂盒,成为HTS的选择。

Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µL of Calbryte™ 520 AM (left), Calbryte™ 590 AM (middle) or Calbryte™ 630 AM (right) in HHBS with 2 mM probenecid were added into the wells, and the cells were incubated at 37 °C for one hour. The dye loading mediums were replaced with 200 µL HHBS, treated with 50 µL of 50 µM ATP, and imaged with a fluorescence microscope (Keyence) using FITC channel (Calbryte™ 520), TRITC channel (Calbryte™ 590) or Texas Red channel (Calbryte™ 630).


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