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新型钙离子荧光探针Calbryte630AM *细胞渗透性*

英文名称:Calbryte™ 630 AM
产品参数
Ex (nm)607Em (nm)624
分子量1234.84溶剂DMSO
存储条件在零下15度以下保存, 避免光照
产品概述

钙测量对于许多生物学研究至关重要。显示结合钙的光谱响应的荧光探针使研究人员能够通过使用荧光显微镜、流式细胞术、荧光光谱和荧光酶标仪来研究细胞内游离钙浓度的变化。 x-Rhod-1 常用作红色荧光钙指示剂。然而,x-Rhod-1 在酯酶水解后仅在活细胞中发出适度的荧光,并且细胞钙反应非常小。 Calbryte™ 630 改变了 x-Rhod-1 细胞负载和钙响应,同时保持 x-Rhod-1 的光谱波长,使其与 Texas Red® 滤光片组兼容。在 CHO 和 HEK 细胞中,Cal-630 AM 的细胞钙反应比 x-Rhod-1 更敏感。 Calbryte 630 的光谱与 FITC、Alexa Fluor® 488 和 GFP 的光谱完全分离,使其成为使用 GFP 细胞系或 FITC/Alexa Fluor® 488 标记抗体进行多重细胞内检测的钙探针。 Calbryte™ 630 是新一代红色荧光指示剂,用于测量细胞内钙。 Calbryte™ 630 AM 显着提高的信号/背景比和细胞内保留特性使其成为最强大的深红色荧光指示剂,用于评估 GPCR 和钙通道靶点以及在活细胞中筛选其激动剂和拮抗剂。与其他染料 AM 细胞负载一样,Calbryte™ 630 AM 酯不发出荧光,一旦进入细胞内,就会被细胞内酯酶水解并被激活。激活的指示剂是一种极性分子,不再能够通过细胞膜自由扩散,基本上被困在细胞内。

点击查看光谱

点击查看实验方案

钙离子篇:时间轴式讲解应用于钙离子检测的探针

 

适用仪器


流式细胞仪  
Ex: 640 nm
Em: 660/20  nm 
通道: APC通道

 


荧光显微镜

Ex: Texas Red 滤波片组
Em: Texas Red 滤波片组
推荐孔板: 黑色透明底板

 


荧光酶标仪

 
Ex: 600 nm
Em: 640 nm
Cutoff: 630 nm
推荐孔板: 黑色透明底板
读取模式: 底读模式/可分液处理
实验方案

实验方案

溶液配制

1.储备溶液配制

Calbryte 630 AM 储备溶液

在无水 DMSO 中制备 2 至 5 mM Calbryte 630 AM 储备溶液。

注意:当在 DMSO 中复溶时,Calbryte 630 AM 是透明、无色的溶液。

2.工作溶液配制

Calbryte 630 AM 工作溶液

2.1实验当天,将 Calbryte 630 AM 溶解在 DMSO 中,或将等份指示剂储备溶液解冻至室温。

2.2在您选择的缓冲液(例如 Hanks 和 Hepes 缓冲液)(货号20011)中用 0.04% Pluronic® F-127 制备 2 至 20 µM Calbryte 630 AM 工作溶液。对于大多数细胞系,建议终浓度为 4-5 μM 的 Calbryte 630 AM。细胞加载所需指示剂的准确浓度必须根据经验确定。

注意:非离子洗涤剂 Pluronic® F-127 有时用于增加 Calbryte 630 AM 的水溶性。可以从百萤购买购买。

注意:如果您的细胞含有有机阴离子转运蛋白,可将丙磺舒 (1-2 mM) 添加到染料工作溶液中(孔中的最终浓度为 0.5-1 mM),以减少脱酯后染料的外漏。各种 ReadiUse 丙磺舒产品,包括水溶性、钠盐和稳定溶液,均可从百萤购买。

 

操作步骤

以下是我们推荐的将Calbryte 630 AM加入活细胞的方案。 该方案仅提供指南,实际应根据您的特定需求进行修改。

a)将细胞在生长培养基中培养过夜。

b)第二天,将 1X Calbryte 630 AM 工作溶液添加到细胞孔板中。

注意:如果您的化合物干扰血清,请在上样前用新鲜的 HHBS 缓冲液替换生长培养基。

c)将载有染料的孔板在细胞培养箱中于 37°C 下孵育 30 至 60 分钟。

注意:孵育染料超过 1 小时可以提高某些细胞系的信号强度。

d)用 HHBS 或您选择的缓冲液(包含阴离子转运蛋白抑制剂,例如 1 mM 丙磺舒,如果适用)替换染料工作溶液,以去除任何多余的探针。

e)将染料加载板在细胞培养箱中孵育约60分钟,然后将板在室温下再孵育15分钟。

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

g)根据需要添加刺激剂,同时使用配备 FITC 滤光片组的荧光显微镜或包含可编程液体处理系统(例如 FDSS、FLIPR 或 FlexStation)的荧光酶标仪在 Ex/Em = 600/640 nm 处测量荧光。

 

产品应用文献

An evolutionary-conserved VPS34-PIKfyve-TRPML1-Myosin II axis regulates the speed of amoeboid cell migration
Authors: Dehio, Philippe and Michard, C{\'e}line and Yam-Puc, Juan Carlos and Mart{\'\i} i L{\'\i}ndez, Adri{\`a}-Arnau and Fabre, Lucien and Schaefer, Thorsten and Wymann, Matthias P and Okkenhaug, Klaus and Soldati, Thierry and Mehling, Matthias and others,
Journal: bioRxiv (2024): 2024--01
 
Heterogeneous signals and soft-geometric network structure in the mouse AV node
Authors: Maltsev, Anna V and Barlas, Yasir and Hazan, Adina T and Zhang, Rui and Goldhaber, Joshua I
Journal: Biophysical Journal (2024): 457a
 
Universal Biomaterial-on-Chip: a versatile platform for evaluating cellular responses on diverse biomaterial substrates
Authors: Atif, Abdul Raouf and Aramesh, Morteza and Carter, Sarah-Sophia and Tenje, Maria and Mestres, Gemma
Journal: Journal of Materials Science: Materials in Medicine (2024): 2
 
Biphasic calcium phosphate recruits Tregs to promote bone regeneration
Authors: Li, Jiaojiao and Zhao, Qin and Wang, Can and Fu, Liangliang and Zhao, Zifan and Tang, Ziqiao and Yin, Chenghu and Wang, Min and Xia, Haibin and others,
Journal: Acta Biomaterialia (2024)
 
Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanism
Authors: Sun, Jie and Tong, Yingying and Jia, Yu and Jia, Xu and Wang, Hua and Chen, Yang and Wu, Jiamin and Jin, Weiyang and Ma, Zheng and Cao, Kai and others,
Journal: Scientific Reports (2023): 6989
 
A Programmable Microfluidic Platform to Monitor Calcium Dynamics in Microglia during Inflammation
Authors: Jones, Caroline and Shebindu, Adam and Kaveti, Durga and Umutoni, Linda and Kirk, Gia and Burton, Michael
Journal: (2023)
 
 
 
Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling
Authors: Buchmann, Sebastian and Enrico, Alessandro and Holzreuter, Muriel Alexandra and Reid, Michael and Zeglio, Erica and Niklaus, Frank and Stemme, G{\"o}ran and Herland, Anna
Journal: Materials Today Bio (2023): 100706
 
Photothermal Excitation of Neurons Using MXene: Cellular Stress and Phototoxicity Evaluation
Authors: Wang, Yingqiao and Hartung, Jane E and Goad, Adam and Preisegger, Mat{\'\i}as A and Chacon, Benjamin and Gold, Michael S and Gogotsi, Yury and Cohen-Karni, Tzahi
Journal: Advanced Healthcare Materials (2023): 2302330

 

参考文献

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)
 
Monosialoganglioside 1 may alleviate neurotoxicity induced by propofol combined with remifentanil in neural stem cells
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945
 
Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781
 
Dexmedetomidine reduces hypoxia/reoxygenation injury by regulating mitochondrial fission in rat hippocampal neurons
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868
 
Di (2-ethylhexyl) phthalate-induced apoptosis in rat INS-1 cells is dependent on activation of endoplasmic reticulum stress and suppression of antioxidant protection
Authors: Sun, Xia and Lin, Yi and Huang, Qiansheng and Shi, Junpeng and Qiu, Ling and Kang, Mei and Chen, Yajie and Fang, Chao and Ye, Ting and Dong, Sijun
Journal: Journal of cellular and molecular medicine (2015): 581--594
 
The effect of mitochondrial calcium uniporter on mitochondrial fission in hippocampus cells ischemia/reperfusion injury
Authors: Zhao, Lantao and Li, Shuhong and Wang, Shilei and Yu, Ning and Liu, Jia
Journal: Biochemical and biophysical research communications (2015): 537--542
 
Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats
Authors: Liang, Nan and Wang, Peng and Wang, Shilei and Li, Shuhong and Li, Yu and Wang, Jinying and Wang, Min
Journal: Journal of Neural Transmission (2014): 593--600
 
Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells
Authors: Li, Qing and Lu, Jiang and Wang, Xianyu and others, undefined
Journal: Neural regeneration research (2014): 2002
 
Fungus induces the release of IL-8 in human corneal epithelial cells, via Dectin-1-mediated protein kinase C pathways.
Authors: Peng, Xu-Dong and Zhao, Gui-Qiu and Lin, Jing and Jiang, Nan and Xu, Qiang and Zhu, Cheng-Cheng and Qu, Jain-Qiu and Cong, Lin and Li, Hui
Journal: International journal of ophthalmology (2014): 441--447
 
Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve--mast cell interaction in a hapten-induced mouse model of atopic dermatitis
Authors: Hagiyama, M and Inoue, T and Furuno, T and Iino, T and Itami, S and Nakanishi, M and Asada, H and Hosokawa, Y and Ito, A
Journal: British Journal of Dermatology (2013): 771--778

 

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