钙离子荧光探针Fluo-4, 五钾盐

	货号	20555	存储条件	在零下15度以下保存
	规格	1 mg	价格	3924
	Ex (nm)	495	Em (nm)	528
	分子量	927.08	溶剂	Water
	产品详细介绍

简要概述

钙的测量对于许多生物学研究至关重要。 荧光探针显示结合钙的光谱响应，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钙浓度的变化。 Fluo-3和Fluo-4最常用于可见光可兴奋的钙指示剂中。 Fluo-4是fluo-3的类似物，其中两个氯取代基被氟取代，这导致488nm处的荧光激发增加，因此荧光信号水平更高。 可以使用贴片移液管或显微注射将细胞物理地加载这些指示物的细胞不渗透盐形式。 这些指标可用于荧光和共聚焦显微镜，流式细胞仪和酶标仪应用。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

使用钙指示剂AM酯类

1.使用钙指示剂AM Esters加载细胞：

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。 它们应该用高质量的无水二甲基亚砜（DMSO）重新配制。 DMSO储备溶液应在-20°C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AM酯加载到活细胞中的方案。 该方案仅提供指南，实际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 AM酯原液。

b）在实验当天，将钙指示剂溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。使用0.04％Pluronic®F-127在您选择的缓冲液（如Hanks和Hepes缓冲液）中制备2至20μM的工作溶液。 对于大多数细胞系，我们建议钙指示剂的最终浓度为4-5 uM。细胞加载所需指标的确切浓度必须根据经验确定。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最小探针浓度。

注意：非离子洗涤剂Pluronic®F-127有时用于增加钙指示剂AM酯的水溶性。

c）如果您的细胞（如CHO细胞）含有有机阴离子转运蛋白，可以将丙磺舒（2-5 mM）或磺吡酮（0.2-0.5 mM）添加到染料工作溶液中（最终浓度为1） 丙磺舒为-2.5 mM，对于磺吡酮为0.1-0.25 mM，以减少去酯化指标的泄漏。

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）将染料加载板室在温度或37℃下孵育20分钟（特别是Fluo-8AM）至2小时，然后将板在室温下再孵育30分钟。

注意1：降低加载温度可能会减少指示符的划分。

注意2：孵育Cal-520 AM超过2小时可以为某些细胞系提供更好的信号强度

f）用HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如1mM丙磺舒，如果适用）替换染料工作溶液，以去除多余的探针。

g）在所需的Ex / Em波长下进行实验（见说明书）。

试剂应用文献

14,15‐epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cells
Authors: Oguro, Ami and Inoue, Takumi and Kudoh, Suguru N and Imaoka, Susumu
Journal: Pharmacology Research & Perspectives (2018): e00428

参考文献

14, 15-epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC 12 and rat hippocampal neuronal cells
Authors: Ami Oguro, Takumi Inoue, Suguru N Kudoh, Susumu Imaoka
Journal: Pharmacology Research & Perspectives (2018): e00428

Succinate, increased in metabolic syndrome, activates GPR91 receptor signaling in urothelial cells
Authors: Abubakr H Mossa, Monica Velasquez Flores, Philippe G Cammisotto, Lysanne Campeau
Journal: Cellular Signalling (2017)

相关产品

钙离子荧光探针Fluo-4, 五钾盐

	货号	20556	存储条件	在零下15度以下保存
	规格	10×50 ug	价格	2328
	Ex (nm)	495	Em (nm)	528
	分子量	927.08	溶剂	Water
	产品详细介绍

简要概述

钙的测量对于许多生物学研究至关重要。 荧光探针显示结合钙的光谱响应，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钙浓度的变化。 Fluo-3和Fluo-4最常用于可见光可兴奋的钙指示剂中。 Fluo-4是fluo-3的类似物，其中两个氯取代基被氟取代，这导致488nm处的荧光激发增加，因此荧光信号水平更高。 可以使用贴片移液管或显微注射将细胞物理地加载这些指示物的细胞不渗透盐形式。 这些指标可用于荧光和共聚焦显微镜，流式细胞仪和酶标仪应用。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

使用钙指示剂AM酯类

1.使用钙指示剂AM Esters加载细胞：

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。 它们应该用高质量的无水二甲基亚砜（DMSO）重新配制。 DMSO储备溶液应在-20°C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AM酯加载到活细胞中的方案。 该方案仅提供指南，实际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 AM酯原液。

b）在实验当天，将钙指示剂溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。使用0.04％Pluronic®F-127在您选择的缓冲液（如Hanks和Hepes缓冲液）中制备2至20μM的工作溶液。 对于大多数细胞系，我们建议钙指示剂的最终浓度为4-5 uM。细胞加载所需指标的确切浓度必须根据经验确定。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最小探针浓度。

注意：非离子洗涤剂Pluronic®F-127有时用于增加钙指示剂AM酯的水溶性。

c）如果您的细胞（如CHO细胞）含有有机阴离子转运蛋白，可以将丙磺舒（2-5 mM）或磺吡酮（0.2-0.5 mM）添加到染料工作溶液中（最终浓度为1） 丙磺舒为-2.5 mM，对于磺吡酮为0.1-0.25 mM，以减少去酯化指标的泄漏。

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）将染料加载板室在温度或37℃下孵育20分钟（特别是Fluo-8AM）至2小时，然后将板在室温下再孵育30分钟。

注意1：降低加载温度可能会减少指示符的划分。

注意2：孵育Cal-520 AM超过2小时可以为某些细胞系提供更好的信号强度

f）用HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如1mM丙磺舒，如果适用）替换染料工作溶液，以去除多余的探针。

g）在所需的Ex / Em波长下进行实验（见说明书）。

试剂应用文献

14,15‐epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cells
Authors: Oguro, Ami and Inoue, Takumi and Kudoh, Suguru N and Imaoka, Susumu
Journal: Pharmacology Research & Perspectives (2018): e00428

参考文献

14, 15-epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC 12 and rat hippocampal neuronal cells
Authors: Ami Oguro, Takumi Inoue, Suguru N Kudoh, Susumu Imaoka
Journal: Pharmacology Research & Perspectives (2018): e00428

Succinate, increased in metabolic syndrome, activates GPR91 receptor signaling in urothelial cells
Authors: Abubakr H Mossa, Monica Velasquez Flores, Philippe G Cammisotto, Lysanne Campeau
Journal: Cellular Signalling (2017)

相关产品

钙离子荧光探针Fluo-8，钠盐

	货号	21086	存储条件	在零下15度以下保存, 避免光照
	规格	1 mg	价格	3924
	Ex (nm)	495	Em (nm)	516
	分子量	796.53	溶剂	Water
	产品详细介绍

简要概述

钙离子荧光探针Fluo-8，钠盐是美国AAT Bioquest生产的用于钙通量测定的试剂，钙测量对于许多生物学研究至关重要。在结合Ca2+后显示光谱响应的荧光探针使研究人员能够使用荧光显微镜，流式细胞仪，荧光光谱和荧光酶标仪来研究细胞内游离Ca2+浓度的变化。在可见光激发钙指示剂中，Fluo-3和Fluo-4最常用。但是，Fluo-3 AM和Fluo-4 AM在酯酶水解后在活细胞中仅适度发荧光，并且需要苛刻的细胞加载条件才能最大化其细胞钙反应。开发Fluo-8®染料可改善细胞负载和钙响应，同时保持便捷的Fluo-3和Fluo-4光谱波长（在〜490 nm处具有最大激发和在〜520 nm处具有最大发射）。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®的亮度是Fluo-4 AM的2倍，是Fluo-3 AM的4倍。AAT Bioquest提供了一套出色的Fluo-8®试剂，具有不同的钙结合亲和力（Fluo-8®：Kd = 389 nM； Fluo-8H：Kd = 232 nM； Fluo-8L：Kd = 1.86 µM； Fluo-8FF ：Kd = 10 µM）。金畔生物是AAT Bioquest 的中国代理商，为您提供最优质的钙离子荧光探针Fluo-8，钠盐。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

操作步骤

1.使用钙指示剂AM Esters加载细胞：

        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。 它们应在使用前用高质量的无水二甲基亚砜（DMSO）重新配制。 DMSO储备溶液应在-20°C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AM酯加载到活细胞中的方案。该方案仅提供指南，实际情况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 AM酯原液。

b）在实验当天，将钙指示剂溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。使用0.02％Pluronic®F-127在您选择的缓冲液（如Hanks和Hepes缓冲液）中制备1至10μM的工作溶液。对于大多数细胞系，我们建议钙指示剂的最终浓度为4-5 uM。 细胞加载所需指标的确切浓度必须根据经验确定。 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最小探针浓度。

注意：非离子洗涤剂Pluronic®F-127有时用于增加钙指示剂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泄漏。 在室温或37°C下用钙指示剂酯孵育细胞20分钟至1小时。

d）在HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如2.5mM丙磺舒，如果适用）中洗涤细胞1-2次以除去过量的探针。

e）在所需的Ex / Em波长下进行实验（见说明书中的表1）。

2.测量细胞内钙响应：

        为了确定溶液的游离钙浓度或单波长钙指示剂的Kd，使用以下等式：[Ca]_free = K_d[F – F_min]/F_max – F]

        其中F是实验钙水平下指示剂的荧光，Fmin是不存在钙时的荧光，Fmax是钙饱和探针的荧光。 解离常数（Kd）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Ca2 +结合和光谱性质在细胞环境中变化非常显着。 细胞内指标的原位校准通常产生显着高于体外测定的Kd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X-100可用于将指示剂暴露于细胞外培养基的受控Ca2 +水平。 说明书中的表1列出了一些钙试剂的Kd值供您参考。

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
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)

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

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
Authors: Tomoyo Tanaka, Mitsuhiro Hoshijima, Junko Sunaga, Takashi Nishida, Mana Hashimoto, Naoya Odagaki, Ryuta Osumi, Taiji Aadachi, Hiroshi Kamioka
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
Authors: Nagendra Babu Thillaiappan, Alap P Chavda, Stephen C Tovey, David L Prole, Colin W Taylor
Journal: Nature Communications (2017): 1505

Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
Authors: Yusuke Hirata, Yuya Morimoto, Eunryel Nam, Shotaro Yoshida, Shoji Takeuchi
Journal: (2017): 13–16

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction
Authors: Qiaoling Sun, Saba Choudhary, Ciaran Mannion, Yair Kissin, Jenny Zilberberg, Woo Y Lee
Journal: Bone (2017)

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx
Authors: ZhongJie Liu, ChangQing Ma, Wei Zhao, QingGuo Zhang, Rui Xu, HongFei Zhang, HongYi Lei, ShiYuan Xu
Journal: Neurochemical Research (2017): 1–14

相关产品

钙离子荧光探针Fluo-8，钾盐

	货号	21087	存储条件	在零下15度以下保存, 避免光照
	规格	1 mg	价格	3924
	Ex (nm)	495	Em (nm)	516
	分子量	877.07	溶剂	Water
	产品详细介绍

简要概述

钙离子荧光探针Fluo-8，钾盐是美国AAT Bioquest生产的用于钙通量测定的试剂，钙测量对于许多生物学研究至关重要。在结合Ca2+后显示光谱响应的荧光探针使研究人员能够使用荧光显微镜，流式细胞仪，荧光光谱和荧光酶标仪来研究细胞内游离Ca2+浓度的变化。在可见光激发钙指示剂中，Fluo-3和Fluo-4最常用。但是，Fluo-3 AM和Fluo-4 AM在酯酶水解后在活细胞中仅适度发荧光，并且需要苛刻的细胞加载条件才能最大化其细胞钙反应。开发Fluo-8®染料可改善细胞负载和钙响应，同时保持便捷的Fluo-3和Fluo-4光谱波长（在〜490 nm处具有最大激发和在〜520 nm处具有最大发射）。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®的亮度是Fluo-4 AM的2倍，是Fluo-3 AM的4倍。AAT Bioquest提供了一套出色的Fluo-8®试剂，具有不同的钙结合亲和力（Fluo-8®：Kd = 389 nM； Fluo-8H：Kd = 232 nM； Fluo-8L：Kd = 1.86 µM； Fluo-8FF ：Kd = 10 µM）。金畔生物是AAT Bioquest 的中国代理商，为您提供最优质的钙离子荧光探针Fluo-8，钾盐。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

操作步骤

1.使用钙指示剂AM Esters加载细胞：

        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。 它们应在使用前用高质量的无水二甲基亚砜（DMSO）重新配制。 DMSO储备溶液应在-20°C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AM酯加载到活细胞中的方案。该方案仅提供指南，实际情况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 AM酯原液。

b）在实验当天，将钙指示剂溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。使用0.02％Pluronic®F-127在您选择的缓冲液（如Hanks和Hepes缓冲液）中制备1至10μM的工作溶液。对于大多数细胞系，我们建议钙指示剂的最终浓度为4-5 uM。 细胞加载所需指标的确切浓度必须根据经验确定。 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最小探针浓度。

注意：非离子洗涤剂Pluronic®F-127有时用于增加钙指示剂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泄漏。 在室温或37°C下用钙指示剂酯孵育细胞20分钟至1小时。

d）在HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如2.5mM丙磺舒，如果适用）中洗涤细胞1-2次以除去过量的探针。

e）在所需的Ex / Em波长下进行实验（见说明书中的表1）。

2.测量细胞内钙响应：

        为了确定溶液的游离钙浓度或单波长钙指示剂的Kd，使用以下等式：[Ca]_free = K_d[F – F_min]/F_max – F]

        其中F是实验钙水平下指示剂的荧光，Fmin是不存在钙时的荧光，Fmax是钙饱和探针的荧光。 解离常数（Kd）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Ca2 +结合和光谱性质在细胞环境中变化非常显着。 细胞内指标的原位校准通常产生显着高于体外测定的Kd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X-100可用于将指示剂暴露于细胞外培养基的受控Ca2 +水平。 说明书中的表1列出了一些钙试剂的Kd值供您参考。

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
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)

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

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
Authors: Tomoyo Tanaka, Mitsuhiro Hoshijima, Junko Sunaga, Takashi Nishida, Mana Hashimoto, Naoya Odagaki, Ryuta Osumi, Taiji Aadachi, Hiroshi Kamioka
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
Authors: Nagendra Babu Thillaiappan, Alap P Chavda, Stephen C Tovey, David L Prole, Colin W Taylor
Journal: Nature Communications (2017): 1505

Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
Authors: Yusuke Hirata, Yuya Morimoto, Eunryel Nam, Shotaro Yoshida, Shoji Takeuchi
Journal: (2017): 13–16

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction
Authors: Qiaoling Sun, Saba Choudhary, Ciaran Mannion, Yair Kissin, Jenny Zilberberg, Woo Y Lee
Journal: Bone (2017)

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx
Authors: ZhongJie Liu, ChangQing Ma, Wei Zhao, QingGuo Zhang, Rui Xu, HongFei Zhang, HongYi Lei, ShiYuan Xu
Journal: Neurochemical Research (2017): 1–14

相关产品

钙离子荧光探针Fluo-8，钠盐

	货号	21088	存储条件	在零下15度以下保存, 避免光照
	规格	10×50 ug	价格	2604
	Ex (nm)	495	Em (nm)	516
	分子量	796.53	溶剂	Water
	产品详细介绍

简要概述

钙离子荧光探针Fluo-8，钠盐是美国AAT Bioquest生产的用于标记钙离子的荧光探针，在许多生物学研究中钙离子的测定非常重要。在与钙离子结合的条件下，荧光探针显现出光谱响应，这使研究者可以通过利用荧光显微镜、流式细胞仪、荧光分光镜和荧光分光仪等来研究细胞内部游离的Ca²⁺浓度的变化。在众多的被可见光激发的钙离子指示剂中，Fluo-3 和 Fluo-4是经常使用的。然而，Fluo-3 AM和Fluo-4 AM在酯酶水解作用下，在活细胞中只产生中度荧光；并且需要更苛刻的细胞载入条件使它们细胞的钙离子响应最大化。Quest Fluo-8 是在保持了Fluo-3和Fluo-4便捷地光谱波长，最大激发光在~490 nm处，最大发生光在~520 nm处的同时，提高了细胞载入和钙离子响应能力。Quest Fluo-8 AM只需要在室温下完成细胞载入，而Fluo-3 AM 和Fluo-4 AM需要在37℃的条件下。此外，Quest Fluo-8 的荧光亮度是Fluo-4 AM的两倍，是Fluo-3 AM的四倍。AAT Bioquest提供一组不同凡响的Quest Fluo-8 试剂，它们与钙离子的结合力都不相同(Quest Fluo-8: Kd = 389 nM; Quest Fluo-8H: Kd = 232 nM; Quest Fluo-8L: Kd = 1.86nM; Quest Fluo-8FF: Kd = 10 nM)。我们也提供多种不同大小的包装以满足你们的特殊要求，例如1 mg; 10×50 ug; 20×50 ug;高通量筛选大包装，不需要您做任何包装剂量上的改变，可直接用于HTS高通量筛选实验分析。金畔生物是AAT Bioquest 的中国代理商，为您提供最优质的钙离子荧光探针。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

操作步骤

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最小染料浓度。

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泄漏。

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Ca ²⁺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Ca ²⁺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 TM，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）以达到最终指示的浓度。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：最大激发波长@ 490 nm; 最大发射@ ~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。
• 最强信号强度：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 _最大 ─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
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)

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

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
Authors: Tomoyo Tanaka, Mitsuhiro Hoshijima, Junko Sunaga, Takashi Nishida, Mana Hashimoto, Naoya Odagaki, Ryuta Osumi, Taiji Aadachi, Hiroshi Kamioka
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
Authors: Nagendra Babu Thillaiappan, Alap P Chavda, Stephen C Tovey, David L Prole, Colin W Taylor
Journal: Nature Communications (2017): 1505

Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
Authors: Yusuke Hirata, Yuya Morimoto, Eunryel Nam, Shotaro Yoshida, Shoji Takeuchi
Journal: (2017): 13–16

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction
Authors: Qiaoling Sun, Saba Choudhary, Ciaran Mannion, Yair Kissin, Jenny Zilberberg, Woo Y Lee
Journal: Bone (2017)

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx
Authors: ZhongJie Liu, ChangQing Ma, Wei Zhao, QingGuo Zhang, Rui Xu, HongFei Zhang, HongYi Lei, ShiYuan Xu
Journal: Neurochemical Research (2017): 1–14

相关产品

钙离子荧光探针Fluo-8，钾盐

	货号	21089	存储条件	在零下15度以下保存, 避免光照
	规格	10×50 ug	价格	2604
	Ex (nm)	495	Em (nm)	516
	分子量	877.07	溶剂	Water
	产品详细介绍

简要概述

钙离子荧光探针Fluo-8，钠盐是美国AAT Bioquest生产的用于标记钙离子的荧光探针，在许多生物学研究中钙离子的测定非常重要。在与钙离子结合的条件下，荧光探针显现出光谱响应，这使研究者可以通过利用荧光显微镜、流式细胞仪、荧光分光镜和荧光分光仪等来研究细胞内部游离的Ca²⁺浓度的变化。在众多的被可见光激发的钙离子指示剂中，Fluo-3 和 Fluo-4是经常使用的。然而，Fluo-3 AM和Fluo-4 AM在酯酶水解作用下，在活细胞中只产生中度荧光；并且需要更苛刻的细胞载入条件使它们细胞的钙离子响应最大化。Quest Fluo-8 是在保持了Fluo-3和Fluo-4便捷地光谱波长，最大激发光在~490 nm处，最大发生光在~520 nm处的同时，提高了细胞载入和钙离子响应能力。Quest Fluo-8 AM只需要在室温下完成细胞载入，而Fluo-3 AM 和Fluo-4 AM需要在37℃的条件下。此外，Quest Fluo-8 的荧光亮度是Fluo-4 AM的两倍，是Fluo-3 AM的四倍。AAT Bioquest提供一组不同凡响的Quest Fluo-8 试剂，它们与钙离子的结合力都不相同(Quest Fluo-8: Kd = 389 nM; Quest Fluo-8H: Kd = 232 nM; Quest Fluo-8L: Kd = 1.86nM; Quest Fluo-8FF: Kd = 10 nM)。我们也提供多种不同大小的包装以满足你们的特殊要求，例如1 mg; 10×50 ug; 20×50 ug;高通量筛选大包装，不需要您做任何包装剂量上的改变，可直接用于HTS高通量筛选实验分析。金畔生物是AAT Bioquest 的中国代理商，为您提供最优质的钙离子荧光探针。

点击查看光谱

钙离子篇：时间轴式讲解应用于钙离子检测的探针

产品说明书

操作步骤

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最小染料浓度。

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泄漏。

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Ca ²⁺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Ca ²⁺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 TM，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）以达到最终指示的浓度。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：最大激发波长@ 490 nm; 最大发射@ ~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。
• 最强信号强度：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 _最大 ─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
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2017)

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

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
Authors: Tomoyo Tanaka, Mitsuhiro Hoshijima, Junko Sunaga, Takashi Nishida, Mana Hashimoto, Naoya Odagaki, Ryuta Osumi, Taiji Aadachi, Hiroshi Kamioka
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
Authors: Nagendra Babu Thillaiappan, Alap P Chavda, Stephen C Tovey, David L Prole, Colin W Taylor
Journal: Nature Communications (2017): 1505

Cells smell on a CMOS: A portable odorant detection system using cell-laden collagen pillars
Authors: Yusuke Hirata, Yuya Morimoto, Eunryel Nam, Shotaro Yoshida, Shoji Takeuchi
Journal: (2017): 13–16

Ex vivo replication of phenotypic functions of osteocytes through biomimetic 3D bone tissue construction
Authors: Qiaoling Sun, Saba Choudhary, Ciaran Mannion, Yair Kissin, Jenny Zilberberg, Woo Y Lee
Journal: Bone (2017)

High Glucose Enhances Isoflurane-Induced Neurotoxicity by Regulating TRPC-Dependent Calcium Influx
Authors: ZhongJie Liu, ChangQing Ma, Wei Zhao, QingGuo Zhang, Rui Xu, HongFei Zhang, HongYi Lei, ShiYuan Xu
Journal: Neurochemical Research (2017): 1–14

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