AAT Bioquest：Calbryte 520 AM

AAT Bioquest產品Calbryte  520 AM（貨號20650）

1. 產品基本信息

Calbryte  520 AM（貨號：20650）是 AAT Bioquest 公司開發的細胞滲透性熒光鈣離子（Ca²⁺）指示劑，專為實時監測活細胞內鈣離子濃度動態變化設計。該產品具備高靈敏度、低細胞毒性及多熒光探針兼容性等特性，廣泛應用于生命科學研究領域。

2. 化學性質與工作原理

2.1 化學結構

Calbryte  520 AM 為乙酰氧基甲酯（AM）衍生物，可通過被動擴散進入細胞。細胞內酯酶水解 AM 保護基團后，釋放出游離的 Calbryte  520 熒光團，其與 Ca??結合后熒光強度顯著增強。

2.2 光譜特性

· 激發波長（λEx）：488 nm（兼容藍光激光）

· 發射波長（λEm）：515–540 nm（綠色熒光范圍）

· 熒光增強特性：與 Ca??結合時熒光信號提升超 100 倍，信噪比（SNR）優異

· 親和力：對 Ca??的解離常數（Kd）約為 1–2 μM，屬于低親和力染料，適用于檢測快速、高幅度的鈣瞬變，避免高親和力染料（如 Fura-2）的飽和問題 。

3. 核心優勢

1. 長時程穩定性：相較于傳統染料 Fluo-4 AM，Calbryte  520 AM 在細胞內保留時間長達 24 小時，無需額外添加有機陰離子轉運抑制劑（如 probenecid）

2. 多通道兼容性：光譜特性支持與紅色熒光探針（如用于肌質網鈣成像的 R-CEPIA1SR）同步使用，實現雙通道鈣信號監測

3. 高靈敏度與低背景：水解后的熒光團與 Ca??結合后產生強熒光信號，背景干擾低，尤其適合活細胞動態成像

4. 應用場景與實驗案例

4.1 心血管研究

· 心肌細胞鈣動力學：用于人誘導多能干細胞分化心肌細胞（hiPSC-CMs）的胞質鈣濃度監測，結合 R-CEPIA1SR 探針同步檢測肌質網鈣釋放，解析心律失常機制（如兒茶酚胺敏感性多形性室速，CPVT）。

· 鈣瞬變與動作電位同步記錄：與膜電位探針 FluoVolt 聯用，實現鈣信號（Calbryte 520）與動作電位（FluoVolt）的雙通道光學映射（100–1000 fps）。

4.2 神經科學

· 神經元光熱刺激研究：在 Ti3C2Tx MXene 材料介導的神經刺激實驗中，實時監測背根神經節（DRG）神經元在光熱刺激下的鈣內流，驗證神經興奮性。

· 鈣信號與凋亡關聯研究：通過巨噬細胞鈣成像，分析炎癥或藥物處理后的鈣響應。

4.3 腫瘤與免疫學

· 腫瘤細胞鈣通量檢測：評估電磁場（如 5 mT 低頻磁場）對腫瘤細胞內鈣濃度的影響，揭示其抑制腫瘤生長的機制 。

· 免疫細胞激活分析：在骨髓中性粒細胞（BMNs）中，監測血小板活化因子（PAF）刺激下的鈣信號動態，研究藥物（如人參皂苷 Rg5）的調控作用 。

4.4 骨與組織工程

· 成骨細胞鈣響應：在電磁治療（EMTT）研究中，檢測骨祖細胞（hOBs）內鈣濃度升高，驗證電場促進骨礦化的機制 。

5. 實驗操作指南

5.1 染色步驟

1. 染料配制：將 Calbryte  520 AM 溶于含 0.04% Pluronic F-127 的緩沖液（如 HBSS 或 KRP-HEPES），終濃度通常為 5 μM。

2. 孵育條件：37°C 避光孵育 30–60 分鐘，隨后室溫平衡 15–30 分鐘以促進酯酶水解。

3. 洗滌與成像：用緩沖液洗滌 3 次后，在熒光顯微鏡或微孔板讀數儀（激發 / 發射：490/525 nm）中觀察信號。

5.2 注意事項

· 需避免長時間光照，以防止熒光探針發生光漂白現象。

· 實驗設計中需設置陰性對照（如未染色細胞）和陽性刺激（如離子霉素），以驗證染料響應 。

6. 與其他鈣染料的對比

總結 

Calbryte  520 AM 憑借長時程穩定性、低背景噪聲和廣泛兼容性，成為活細胞鈣成像的優選工具。其在心血管疾病機制研究、神經興奮性調控及腫瘤治療策略開發等領域展現出顯著應用潛力。更多產品問題，歡迎咨詢AAT Bioquest中國區總代理，艾美捷科技，www.zihai029.com

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