AAT Bioquest產(chǎn)品Calbryte  520 AM（貨號(hào)20650）

1. 產(chǎn)品基本信息

Calbryte  520 AM（貨號(hào)：20650）是 AAT Bioquest 公司開(kāi)發(fā)的細(xì)胞滲透性熒光鈣離子（Ca²⁺）指示劑，專為實(shí)時(shí)監(jiān)測(cè)活細(xì)胞內(nèi)鈣離子濃度動(dòng)態(tài)變化設(shè)計(jì)。該產(chǎn)品具備高靈敏度、低細(xì)胞毒性及多熒光探針兼容性等特性，廣泛應(yīng)用于生命科學(xué)研究領(lǐng)域。

2. 化學(xué)性質(zhì)與工作原理

2.1 化學(xué)結(jié)構(gòu)

Calbryte  520 AM 為乙酰氧基甲酯（AM）衍生物，可通過(guò)被動(dòng)擴(kuò)散進(jìn)入細(xì)胞。細(xì)胞內(nèi)酯酶水解 AM 保護(hù)基團(tuán)后，釋放出游離的 Calbryte  520 熒光團(tuán)，其與 Ca??結(jié)合后熒光強(qiáng)度顯著增強(qiáng)。

2.2 光譜特性

· 激發(fā)波長(zhǎng)（λEx）：488 nm（兼容藍(lán)光激光）

· 發(fā)射波長(zhǎng)（λEm）：515–540 nm（綠色熒光范圍）

· 熒光增強(qiáng)特性：與 Ca??結(jié)合時(shí)熒光信號(hào)提升超 100 倍，信噪比（SNR）優(yōu)異

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

3. 核心優(yōu)勢(shì)

1. 長(zhǎng)時(shí)程穩(wěn)定性：相較于傳統(tǒng)染料 Fluo-4 AM，Calbryte  520 AM 在細(xì)胞內(nèi)保留時(shí)間長(zhǎng)達(dá) 24 小時(shí)，無(wú)需額外添加有機(jī)陰離子轉(zhuǎn)運(yùn)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(zhì)網(wǎng)鈣成像的 R-CEPIA1SR）同步使用，實(shí)現(xiàn)雙通道鈣信號(hào)監(jiān)測(cè)

3. 高靈敏度與低背景：水解后的熒光團(tuán)與 Ca??結(jié)合后產(chǎn)生強(qiáng)熒光信號(hào)，背景干擾低，尤其適合活細(xì)胞動(dòng)態(tài)成像

4. 應(yīng)用場(chǎng)景與實(shí)驗(yàn)案例

4.1 心血管研究

· 心肌細(xì)胞鈣動(dòng)力學(xué)：用于人誘導(dǎo)多能干細(xì)胞分化心肌細(xì)胞（hiPSC-CMs）的胞質(zhì)鈣濃度監(jiān)測(cè)，結(jié)合 R-CEPIA1SR 探針同步檢測(cè)肌質(zhì)網(wǎng)鈣釋放，解析心律失常機(jī)制（如兒茶酚胺敏感性多形性室速，CPVT）。

· 鈣瞬變與動(dòng)作電位同步記錄：與膜電位探針 FluoVolt 聯(lián)用，實(shí)現(xiàn)鈣信號(hào)（Calbryte 520）與動(dòng)作電位（FluoVolt）的雙通道光學(xué)映射（100–1000 fps）。

4.2 神經(jīng)科學(xué)

· 神經(jīng)元光熱刺激研究：在 Ti3C2Tx MXene 材料介導(dǎo)的神經(jīng)刺激實(shí)驗(yàn)中，實(shí)時(shí)監(jiān)測(cè)背根神經(jīng)節(jié)（DRG）神經(jīng)元在光熱刺激下的鈣內(nèi)流，驗(yàn)證神經(jīng)興奮性。

· 鈣信號(hào)與凋亡關(guān)聯(lián)研究：通過(guò)巨噬細(xì)胞鈣成像，分析炎癥或藥物處理后的鈣響應(yīng)。

4.3 腫瘤與免疫學(xué)

· 腫瘤細(xì)胞鈣通量檢測(cè)：評(píng)估電磁場(chǎng)（如 5 mT 低頻磁場(chǎng)）對(duì)腫瘤細(xì)胞內(nèi)鈣濃度的影響，揭示其抑制腫瘤生長(zhǎng)的機(jī)制 。

· 免疫細(xì)胞激活分析：在骨髓中性粒細(xì)胞（BMNs）中，監(jiān)測(cè)血小板活化因子（PAF）刺激下的鈣信號(hào)動(dòng)態(tài)，研究藥物（如人參皂苷 Rg5）的調(diào)控作用 。

4.4 骨與組織工程

· 成骨細(xì)胞鈣響應(yīng)：在電磁治療（EMTT）研究中，檢測(cè)骨祖細(xì)胞（hOBs）內(nèi)鈣濃度升高，驗(yàn)證電場(chǎng)促進(jìn)骨礦化的機(jī)制 。

5. 實(shí)驗(yàn)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(jìn)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(shù)儀（激發(fā) / 發(fā)射：490/525 nm）中觀察信號(hào)。

5.2 注意事項(xiàng)

· 需避免長(zhǎng)時(shí)間光照，以防止熒光探針發(fā)生光漂白現(xiàn)象。

· 實(shí)驗(yàn)設(shè)計(jì)中需設(shè)置陰性對(duì)照（如未染色細(xì)胞）和陽(yáng)性刺激（如離子霉素），以驗(yàn)證染料響應(yīng) 。

6. 與其他鈣染料的對(duì)比

總結(jié) 

Calbryte  520 AM 憑借長(zhǎng)時(shí)程穩(wěn)定性、低背景噪聲和廣泛兼容性，成為活細(xì)胞鈣成像的優(yōu)選工具。其在心血管疾病機(jī)制研究、神經(jīng)興奮性調(diào)控及腫瘤治療策略開(kāi)發(fā)等領(lǐng)域展現(xiàn)出顯著應(yīng)用潛力。更多產(chǎn)品問(wèn)題，歡迎咨詢AAT Bioquest中國(guó)區(qū)總代理，艾美捷科技，m.zihai029.com

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