AAT Bioquest产品Calbryte 520 AM（货号20650）
1. 产品基本信息
Calbryte 520 AM（货号：20650）是 AAT Bioquest 公司开发的细胞渗透性荧光钙离子（Ca2+）指示剂，专为实时监测活细胞内钙离子浓度动态变化设计。该产品具备高灵敏度、低细胞毒性及多荧光探针兼容性等特性，广泛应用于生命科学研究领域。
2. 化学性质与工作原理
2.1 化学结构
Calbryte 520 AM 为乙酰氧基甲酯（AM）衍生物，可通过被动扩散进入细胞。细胞内酯酶水解 AM 保护基团后，释放出游离的 Calbryte 520 荧光团，其与 Ca??结合后荧光强度显著增强。
2.2 光谱特性
· 激发波长（λEx）：488 nm（兼容蓝光激光）
· 发射波长（λEm）：515–540 nm（绿色荧光范围）
· 荧光增强特性：与 Ca??结合时荧光信号提升超 100 倍，信噪比（SNR）优异
· 亲和力：对 Ca??的解离常数（Kd）约为 1–2 μM，属于低亲和力染料，适用于检测快速、高幅度的钙瞬变，避免高亲和力染料（如 Fura-2）的饱和问题 。
3. 核心优势
1. 长时程稳定性：相较于传统染料 Fluo-4 AM，Calbryte 520 AM 在细胞内保留时间长达 24 小时，无需额外添加有机阴离子转运抑制剂（如 probenecid）
2. 多通道兼容性：光谱特性支持与红色荧光探针（如用于肌质网钙成像的 R-CEPIA1SR）同步使用，实现双通道钙信号监测
3. 高灵敏度与低背景：水解后的荧光团与 Ca??结合后产生强荧光信号，背景干扰低，尤其适合活细胞动态成像
4. 应用场景与实验案例
4.1 心血管研究
· 心肌细胞钙动力学：用于人诱导多能干细胞分化心肌细胞（hiPSC-CMs）的胞质钙浓度监测，结合 R-CEPIA1SR 探针同步检测肌质网钙释放，解析心律失常机制（如儿茶酚胺敏感性多形性室速，CPVT）。
· 钙瞬变与动作电位同步记录：与膜电位探针 FluoVolt 联用，实现钙信号（Calbryte 520）与动作电位（FluoVolt）的双通道光学映射（100–1000 fps）。
4.2 神经科学
· 神经元光热刺激研究：在 Ti3C2Tx MXene 材料介导的神经刺激实验中，实时监测背根神经节（DRG）神经元在光热刺激下的钙内流，验证神经兴奋性。
· 钙信号与凋亡关联研究：通过巨噬细胞钙成像，分析炎症或药物处理后的钙响应。
4.3 肿瘤与免疫学
· 肿瘤细胞钙通量检测：评估电磁场（如 5 mT 低频磁场）对肿瘤细胞内钙浓度的影响，揭示其抑制肿瘤生长的机制 。
· 免疫细胞激活分析：在骨髓中性粒细胞（BMNs）中，监测血小板活化因子（PAF）刺激下的钙信号动态，研究药物（如人参皂苷 Rg5）的调控作用 。
4.4 骨与组织工程
· 成骨细胞钙响应：在电磁治疗（EMTT）研究中，检测骨祖细胞（hOBs）内钙浓度升高，验证电场促进骨矿化的机制 。
5. 实验操作指南
5.1 染色步骤
1. 染料配制：将 Calbryte 520 AM 溶于含 0.04% Pluronic F-127 的缓冲液（如 HBSS 或 KRP-HEPES），终浓度通常为 5 μM。
2. 孵育条件：37°C 避光孵育 30–60 分钟，随后室温平衡 15–30 分钟以促进酯酶水解。
3. 洗涤与成像：用缓冲液洗涤 3 次后，在荧光显微镜或微孔板读数仪（激发 / 发射：490/525 nm）中观察信号。
5.2 注意事项
· 需避免长时间光照，以防止荧光探针发生光漂白现象。
· 实验设计中需设置阴性对照（如未染色细胞）和阳性刺激（如离子霉素），以验证染料响应 。
6. 与其他钙染料的对比
| 特性 | Calbryte 520 AM | Fluo-4 AM | Rhod-2 AM |
| 亲和力（Kd） | ~1–2 uM（低） | ~345 nM（高） | ~1 uM（中等） |
| 保留时间 | 长达 24 小时（无需 probenecid） | 需 probenecid 延长保留 | 中等 |
| 多通道兼容性 | 兼容红色探针（如 R-CEPIA） | 需避免光谱重叠 | 适合单通道成像 |
| 典型应用场景 | 快速钙瞬变、长时程实验 | 稳态钙浓度检测 | 线粒体钙监测 |
总结
Calbryte 520 AM 凭借长时程稳定性、低背景噪声和广泛兼容性，成为活细胞钙成像的优选工具。其在心血管疾病机制研究、神经兴奋性调控及肿瘤治疗策略开发等领域展现出显著应用潜力。更多产品问题，欢迎咨询AAT Bioquest中国区总代理，艾美捷科技，www.amyjet.com
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