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

作者：admin信息來源：達科為日期：2018年01月30日打印字體：大中小

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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⁺第二信使學說。細胞內Ca2⁺的分布與轉移是形成Ca2⁺信號的基礎，因此監(jiān)測Ca2⁺的動態(tài)變化是研究G蛋白偶聯受體（GPCR）有效刺激劑的重要手段。

AAT Bioquest 提供各種優(yōu)質鈣離子檢測探針

1 Fluo8

Fluo-3和Fluo-4是常用的可見光激發(fā)的Ca2⁺指示劑，然而Fluo-3^AM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來加強熒光強度。AAT Bioquest^?Fluo-8改善了二者的loading condition和熒光強度問題，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入到細胞內；二是AM基團保證了Calbryte? 熒光探針在沒有進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)光同樣是常見的499nm。

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

這里跟大家提一個名詞，p-糖蛋白，p-glycoprotein 1 ，P-gp)，在很多細胞里比如Hella，P-gp扮演著ATP依賴的離子泵將很多小分子泵出細胞外，當然進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。有的會加入丙磺舒（probenecid），一種通道抑制劑來減少這種泄露。但是這種物質的添加并不理想，有報道TRPV2受體會被其激活而TAS2R 受體又會被其抑制，因此會給研究帶來不可預知的可變因素。

而Calbryte? dyes卻能夠通過平衡離子電量來擺脫窘境。進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probenecid、無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).