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    AAT Bioquest?鈣離子檢測探針,39.7分的文章,想發(fā)高分的看過(guò)來(lái)

    作者:admin信息來(lái)源:達科為日期:2018年01月30日打印字體:  
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    70年代初,在Ca2+受體蛋白-鈣調素(CaM)的發(fā)現極其功能的研究基礎上提出Ca2+第二信使學(xué)說(shuō)。細胞內Ca2+的分布與轉移是形成Ca2+信號的基礎,因此監測Ca2+的動(dòng)態(tài)變化是研究G蛋白偶聯(lián)受體(GPCR)有效刺激劑的重要手段。


    AAT Bioquest 提供各種優(yōu)質(zhì)鈣離子檢測探針

    1   Fluo8

    Fluo-3和Fluo-4是常用的可見(jiàn)光激發(fā)的Ca2+指示劑,然而Fluo-3AM和Fluo-4 在體內結合鈣離子后的熒光強度不高,需要比較苛刻的loading condition來(lái)加強熒光強度。AAT Bioquest?Fluo-8改善了二者的loading condition和熒光強度問(wèn)題,比如Fluo-8只需要在室溫孵育,而Fluo-3和Fluo-4需要37度水浴,另外Fluo-8的熒光強度是Fluo-4的兩倍,是Fluo-3的4倍,另外Fluo-8依舊保持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



    為什么Calbryte?系列能夠提高信噪比


    首先Calbryte?系列被偶聯(lián)了乙酰氧基甲酯( acetoxymethyl ,AM),意義有二:一是利用AM的親酯性使得探針能夠順利進(jìn)入到細胞內;二是AM基團保證了Calbryte? 熒光探針在沒(méi)有進(jìn)入細胞之前的非活性以及非熒光的特性,這樣就能減少非特異性信號,提高信噪比。

     

         

    為什么Calbryte?系列熒光強度更強?



    因為該系列染料有更高的量子產(chǎn)率,是Fluo-3 or Fluo-4.的3倍,什么意思呢,就是吸收同樣數目的光子,Calbryte?系列能發(fā)出的熒光強度是它們的3倍,而且激發(fā)光同樣是常見(jiàn)的499nm。

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



    這里跟大家提一個(gè)名詞,p-糖蛋白,p-glycoprotein 1 ,P-gp),在很多細胞里比如Hella,P-gp扮演著(zhù)ATP依賴(lài)的離子泵將很多小分子泵出細胞外,當然進(jìn)入細胞的鈣離子探針也免不了這種遭遇。這就導致被排到細胞外的探針會(huì )結合胞外的離子,造成背景值增高而胞內的目的信號變弱,信噪比降低。有的會(huì )加入丙磺舒(probenecid),一種通道抑制劑來(lái)減少這種泄露。但是這種物質(zhì)的添加并不理想,有報道TRPV2受體會(huì )被其激活而TAS2R 受體又會(huì )被其抑制,因此會(huì )給研究帶來(lái)不可預知的可變因素。

    而Calbryte? dyes卻能夠通過(guò)平衡離子電量來(lái)擺脫窘境。進(jìn)入細胞后一旦被水解,便形成帶有大量負電荷的親水性分子大大降低了這種泄露。

     

    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? 提供無(wú)probenecid、無(wú)需洗滌的鈣離子檢測試劑盒,成為HTS的選擇。

    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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