钙离子荧光探针Fluo8H钠盐

钙离子荧光探针Fluo-8H, 钠盐是美国AAT Bioquest生产的用于标记钙离子的荧光探针，钙的测量对于许多生物学研究至关重要。荧光探针显示结合Ca²⁺后的光谱响应，使研究人员能够通过荧光显微镜，流式细胞仪，荧光光谱和荧光酶标仪研究细胞内游离Ca²⁺浓度的变化。Fluo-3和Fluo-4常用于可见光可兴奋的钙指示剂中。然而，Fluo-3 AM和Fluo-4 AM在酯酶水解后仅在活细胞中发生中度荧光，并且需要苛刻的细胞负载条件以大化其细胞钙响应。Fluo-8®染料的开发是为了细胞负载和钙响应，同时保持方便的Fluo-3和Fluo-4光谱波长，大激发波长为~490 nm，大发射波长为~520 nm。Fluo-8®AM仅需要室温，而Fluo-3 AM和Fluo-4 AM需要37℃的细胞负载。此外，Fluo-8®比Fluo-4 AM亮2倍，比Fluo-3 AM亮4倍。AAT Bioquest提供一系列具有不同钙结合亲和力的优异Fluo-8®试剂（Fluo-8®：Kd = 389 nM; Fluo-8H：Kd = 232 nM; Fluo-8L：Kd =1.86μM; Fluo-8FF ：Kd =10μM）。我们还提供多种包装尺寸，以满足您的特殊需求，例如1毫克; 10x50μg; 20x50μg; HTS包装，无需额外包装费用。百萤生物是AAT Bioquest 的中国代理商，为您提供优质的钙离子荧光探针。

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钙离子篇：时间轴式讲解应用于钙离子检测的探针

使用Fluo-8®AM酯类

1.使用Fluo-8®AM酯：

AM酯是非极性酯，其易于穿过活细胞膜，并且通过活细胞内的细胞酯酶快速水解。AM酯广泛用于非侵入性地将各种极性荧光探针装载到活细胞中。但是，使用AM酯时必须小心，因为它们易于水解，特别是在溶液中。它们应在使用前重新配制成高质量的无水二甲基亚砜（DMSO）。DMSO储备溶液可以在-20℃下干燥储存并避光。在这些条件下，AM酯应稳定数月。

以下是我们推荐的将Fluo-8®AM酯加入活细胞的方案。该协议仅提供指南，应根据您的具体需求进行修改。

a）在高质量无水DMSO中制备2至5 mM Fluo-8®AM酯原液。

b）在实验当天，将Fluo-8® 溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。在Hanks和Hepes缓冲液（HHBS）或0.02％Pluronic®F-127的缓冲液中制备1至10μM的工作溶液。对于大多数细胞系，建议使用浓度范围为4-5 uM的Fluo-8®试剂。细胞加载所需指示剂的确切浓度必须凭经验确定。为避免因过载和潜在染料毒性引起的任何伪影，建议使用可产生足够信号强度的小染料浓度。

注意：非离子洗涤剂Pluronic®F-127有时用于增加Fluo-8®AM 酯的水溶性。 

c）如果您的细胞含有有机阴离子转运蛋白，可以在细胞培养基中加入丙磺舒（1-2.5 mM）或磺吡酮（0.1-0.25 mM），以减少脱酯化指标的渗漏。

d）将等体积的染料工作溶液（来自步骤b或c）加入细胞板中。

e）在细胞培养箱中孵育或染料装载板室温下为20分钟至一小时。

f）用HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如2.5 mM丙磺舒，如果适用）替换染料工作溶液，以去除多余的探针。

g）运行用Ex / Em比值=490/525纳米

 

使用Screen Quest Fluo-8 NW钙测定试剂盒进行HTS应用

        可以通过直接测量受体介导的cAMP积累或细胞内Ca²⁺浓度的变化来检测GPCR活化。通过Gq偶联的GPCR靶标产生细胞内Ca²⁺的增加可以使用Fluo-8®试剂和荧光酶标仪的组合进行测量。荧光成像板读取器（例如，FLIPR，FDSS或BMG NovoStar）具有冷却的CCD相机成像系统，其同时收集来自微孔板（96孔和384孔）的每个孔的信号。这些读板器可以以亚秒的间隔读取，这使得能够捕获响应的动力学，并且具有可以被编程用于连续液体添加的集成移液器。除了对GPCR靶标的强大应用外，我们的Screen Quest Fluo-8钙测定试剂盒还可用于表征钙离子通道和筛选钙离子通道靶向化合物。

图1.使用Screen Quest Fluo-8 NW测定试剂盒和Fluo-4 NW测定试剂盒在HEK-293细胞中测量卡巴胆碱剂量反应。 将HEK-293细胞以40,000个细胞/100μL/孔接种过夜，置于96孔黑色壁/透明底板中。除去生长培养基，并将细胞分别与100μL的Screen Quest Fluo 8-NW钙测定试剂盒和Fluo-4 NW试剂盒（根据制造商的说明书）在室温下温育1小时。通过NOVOstar（BMG LabTech）添加卡巴胆碱（25μL/孔）以达到终指示的浓度。Fluo-8 NW 的EC ₅₀约为1.2 uM。

与基于Fluo-3或Fluo-4的其他商业钙测定试剂盒相比，我们的Screen Quest 钙测定试剂盒具有以下HTS应用优势：

• 广泛的应用：与GPCR和钙通道目标一起使用。
• 方便的光谱波长：大激发波长@ 490 nm; 大发射@ ~514 nm。
• 灵活的染料加载：室温下的染料加载（而不是Fluo-4 AM所需的37ºC）。
• 无需清洗，无淬火干扰您的目标。
• 强大的性能：使用Fluo-4 AM或Fluo-3 AM无法进行钙分析。
• 强信号强度：比Fluo-4 AM亮2倍;比Fluo-3 AM亮4倍。

使用Fluo-8® 盐

        钙校准可以通过测量具有精确已知的游离Ca ²⁺浓度的溶液中的指示剂的盐形式（荧光酶标仪中25至50μM）的荧光强度来进行。可以基于30mM MOPS EGTA Ca ²⁺缓冲液使用校准溶液。通常，水含有微量的钙离子。强烈建议使用30 mM MOPS + 100 mM KCl，pH 7.2作为缓冲系统。可以简单地制备如下所列的0和39μM钙原液，这两种溶液用于制备不同Ca ²⁺浓度的连续溶液

A.0μM钙：30mM MOPS + 100mM KCl，pH 7.2缓冲液+ 10mM EGTA

B.39μM钙：30mM MOPS + 100mM KCl，pH 7.2缓冲液+ 10mM EGTA + 10mM CaCl ₂

 

为了确定溶液的游离钙浓度或 单波长钙指示剂的K _d，使用以下等式：

的[Ca] _游离 = K _d [F─˚F _分钟 ] / F _大 ─F]

其中F是特定实验钙水平下指示剂的荧光强度，F _min是不存在钙时的荧光强度，F _max是钙饱和探针的荧光强度。

 

解离常数（K _d）是探针对钙的亲和力的量度。与校准溶液相比，荧光指示剂的钙结合和光谱性质在细胞环境中变化非常显着。细胞内指标的原位反应校准通常产生显着高于体外测定的K _d值。通过在离子载体如A-23187,4-溴A-23187和离子霉素存在下将加载的细胞暴露于受控的Ca ²⁺缓冲液来进行原位校准。或者，细胞透化剂如洋地黄皂苷或Triton®X-100可用于将指示剂暴露于受控Ca.²⁺水平的细胞外培养基。

 

试剂应用文献

AMPA receptors in the synapse turnover by monomer diffusion
Authors: Morise, Jyoji and Suzuki, Kenichi GN and Kitagawa, Ayaka and Wakazono, Yoshihiko and Takamiya, Kogo and Tsunoyama, Taka A and Nemoto, Yuri L and Takematsu, Hiromu and Kusumi, Akihiro and Oka, Shogo
Journal: Nature communications (2019): 1--18

Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling
Authors: Feng, Shengjie and Dang, Shangyu and Han, Tina Wei and Ye, Wenlei and Jin, Peng and Cheng, Tong and Li, Junrui and Jan, Yuh Nung and Jan, Lily Yeh and Cheng, Yifan
Journal: Cell Reports (2019): 567--579

Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium
Authors: Fukushima, Tsuyoshi and Tanaka, Yosuke and Hamey, Fiona K and Chang, Chih-Hsiang and Oki, Toshihiko and Asada, Shuhei and Hayashi, Yasutaka and Fujino, Takeshi and Yonezawa, Taishi and Takeda, Reina and others
Journal: Cell Reports (2019): 4144--4158

Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor
Authors: Grossert, Aless and ra and Mehrjardi, Narges Zare and Bailey, Sarah J and Lindsay, Mark A and Hescheler, Jürgen and Saric, Tomo and Teusch, Nicole
Journal: Cells (2019): 1139

MRGPRX4 is a bile acid receptor for human cholestatic itch
Authors: Yu, Huasheng and Zhao, Tianjun and Liu, Simin and Wu, Qinxue and Johnson, Omar and Wu, Zhaofa and Zhuang, Zihao and Shi, Yaocheng and Peng, Luxin and He, Renxi and others
Journal: eLife (2019): e48431

P2Y6 signaling in alveolar macrophages prevents leukotriene-dependent type 2 allergic lung inflammation
Authors: Nagai, Jun and Balestrieri, Barbara and Fanning, Laura B and Kyin, Timothy and Cirka, Haley and Lin, Junrui and Idzko, Marco and Zech, Andreas and Kim, Edy Y and Brennan, Patrick J and others
Journal: The Journal of clinical investigation (2019)

Hyperglycaemia disrupts conducted vasodilation in the resistance vasculature of db/db mice
Authors: Lemmey, Hamish AL and Ye, Xi and Ding, Hong C and Triggle, Christopher R and Garland, Christopher J and Dora, Kim A
Journal: Vascular pharmacology (2018): 29--35

Methionine and valine activate the mammalian target of rapamycin complex 1 pathway through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) and intracellular Ca2+ in bovine mammary epithelial cells
Authors: Zhou, Y and Zhou, Z and Peng, J and Loor, Juan J
Journal: Journal of dairy science (2018): 11354--11363

TRPA1-dependent reversible opening of tight junction by natural compounds with an $\alpha$, $\beta$-unsaturated moiety and capsaicin
Authors: Kanda, Yusuke and Yamasaki, Youhei and Sasaki-Yamaguchi, Yoshie and Ida-Koga, Noriko and Kamisuki, Shinji and Sugawara, Fumio and Nagumo, Yoko and Usui, Takeo
Journal: Scientific reports (2018): 1--13

A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels
Authors: Menegon, A and Pitassi, S and Mazzocchi, N and Redaelli, L and Rizzetto, R and Roll and JF and Poli, C and Imberti, M and Lanati, A and Grohovaz, F
Journal: Scientific Reports (2017)

Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage
Authors: Gong, Xiaoyuan and Xie, Wenbin and Wang, Bin and Gu, Lingchuan and Wang, Fuyou and Ren, Xiang and Chen, Cheng and Yang, Liu
Journal: Scientific reports (2017): 17093

Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks
Authors: Ishii, Masaaki and Rohrer, Bärbel
Journal: Cell Death Discovery (2017): 16071

Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks
Authors: Ishii, Masaaki and Rohrer, Bärbel
Journal: Cell Death Discovery (2017): 16071

High-throughput screen detects calcium signaling dysfunction in typical sporadic autism spectrum disorder
Authors: Schmunk, Galina and Nguyen, Rachel L and Ferguson, David L and Kumar, Kenny and Parker, Ian and Gargus, J Jay
Journal: Scientific Reports (2017): 40740

 

参考文献

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from βTC-3 pancreatic cells: Evaluation of structure-dependent biological activity
Authors: Anna Drzazga, Agata Sowińska, Agnieszka Krzemińska, Andrzej Okruszek, Piotr Paneth, Maria Koziolkiewicz, Edyta Gendaszewska-Darmach
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A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels
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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage
Authors: Xiaoyuan Gong, Wenbin Xie, Bin Wang, Lingchuan Gu, Fuyou Wang, Xiang Ren, Cheng Chen, Liu Yang
Journal: Scientific reports (2017): 17093

Analysis of Ca2+ response of osteocyte network by three-dimensional time-lapse imaging in living bone
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Journal: Journal of Bone and Mineral Metabolism (2017): 1--10

Aryl-and alkyl-phosphorus-containing flame retardants induced mitochondrial impairment and cell death in Chinese hamster ovary (CHO-k1) cells
Authors: Chao Huang, Na Li, Shengwu Yuan, Xiaoya Ji, Mei Ma, Kaifeng Rao, Zijian Wang
Journal: Environmental Pollution (2017): 775--786

Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks
Authors: Masaaki Ishii, Bärbel Rohrer
Journal: Cell Death Discovery (2017): 16071

Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions
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Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
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Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction
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High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx
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