Calbryte 520 AM是一种具有卓越亮度与光稳定性的绿色荧光钙离子指示剂，能够灵敏检测活细胞中的钙离子通量，适用于GPCR筛选及钙信号传导研究。
• 高品质试剂：严格遵循最高生产标准，确保实验结果稳定可靠
• 卓越性能：经多种实验条件验证具有可靠效能
• 广泛应用：适用于各类研究及诊断检测场景
• 低背景荧光：与Ca²⁺结合后显著增强荧光信号，能有效降低背景干扰，提高检测灵敏度。
• 细胞膜通透性： 疏水性AM酯可被动穿透细胞膜，被胞内酯酶水解后形成带负电荷的荧光染料，实现简单负载。
钙离子检测在生命科学研究中具有关键作用。基于荧光探针与钙离子结合的光谱响应特性，研究人员可通过荧光显微镜、流式细胞仪、荧光光谱和荧光酶标仪等技术精确监测细胞内游离钙离子浓度的动态变化。自1989年Fluo-3问世以来，后续开发的Fluo-4、Fluo-8和Cal-520系列探针虽提升了信背比，其仍存在显著局限：突出问题在于使用Fluo-4 AM进行细胞内钙检测时需添加丙磺舒以阻止染料外漏——这一限制与其前代产品Fluo-3相同。对丙磺舒的依赖会影响检测可靠性，因为该试剂已知会对细胞产生复杂的干扰效应，可能导致结果偏差。
Calbryte 520 AM正是针对这些挑战而研发的新一代解决方案。这种新型细胞渗透性钙离子指示剂以无荧光、非活性形式进入细胞后，经细胞内酯酶水解AM酯基激活，转化为具有钙响应能力的极性分子。水解产物因无法跨膜而高效滞留于细胞内，彻底消除了对丙磺舒的需求。当与钙离子结合时，Calbryte 520能产生亮度极高且信噪比卓越的荧光信号。值得注意的是，该指示剂与Fluo-4具有完全相同的激发和发射波长，可直接兼容现有Fluo-4检测方案。凭借其增强的信号质量、改进的细胞内滞留能力以及无需丙磺舒的操作优势，Calbryte 520 AM已成为评估GPCR与钙离子通道靶点、筛选活细胞内相关激动剂与拮抗剂时最可靠的钙离子指示剂。
光谱性质
激发波长 (nm)：493
发射波长 (nm)：515
荧光量子产率：0.75
适用仪器
流式细胞仪
Ex: 488 nm
Em： 530/30 nm
通道: FITC通道
荧光显微镜
Ex: FITC 滤波片组
Em： FITC 滤波片组
推荐孔板: 黑色透明底板
荧光酶标仪
Ex: 490 nm
Em： 525 nm
Cutoff： 515 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
储备溶液的配制
除非另有说明，所有未使用的储备液应在配制后分装为单次使用量，并保存于-20°C。避免反复冻融。
Calbryte 520 AM 储备溶液
1. 使用高质量无水DMSO配制2-5 mM的Calbryte 520 AM储备液。
注意：Calbryte 520 AM溶于DMSO后为无色透明溶液。
工作溶液配制
Calbryte520 AM 工作溶液
1. 实验当天，将Calbryte520 AM溶解于DMSO或将储备液 aliquot 解冻至室温。
2. 在所选缓冲液（如Hanks和Hepes缓冲液）中加入0.04% Pluronic® F-127，配制2-20 µM的Calbryte 520 AM工作液。对于多数细胞系，推荐终浓度为4-5 μM。具体染料加载浓度需根据实验条件优化。
注意：
1. 非离子型表面活性剂Pluronic® F-127可增强Calbryte 520 AM的水溶性。
2. 若细胞存在有机阴离子转运体，可在工作液中加入1-2 mM丙磺舒（孔板终浓度0.5-1 mM）以减少水解后染料的泄漏。
实验操作流程
以下为活细胞加载AM酯的推荐方案，请根据实际需求调整。
1. 将细胞于生长培养基中孵育过夜。
2. 次日向细胞板中加入1X Calbryte 520 AM工作液。
注意：若待测化合物与血清存在干扰，加载前需更换为新鲜HHBS缓冲液。
3. 将加载染料的细胞板置于37°C培养箱孵育1-2小时。
4. 移除工作液，更换为HHBS或含阴离子转运抑制剂（如1 mM丙磺舒）的缓冲液以清除多余探针。
5. 加入刺激剂后，立即使用配备FITC滤光片的荧光显微镜或者具备程序化液体处理系统的荧光酶标仪（如FDSS、FLIPR或FlexStation）Ex/Em = 490/525 nm，截止波长515 nm检测荧光信号。
Calbryte 520 AM is a green-fluorescent calcium indicator with superior brightness and photostability that enables sensitive detection of calcium flux in live cells for GPCR screening and calcium signaling studies.
• High-quality Reagent: Manufactured to the highest standards for consistent results
• Reliable Performance: Proven effectiveness across multiple experimental conditions
• Versatile Applications: Suitable for a wide range of research and diagnostic applications
• Low background fluorescence: Its fluorescence signal increases dramatically upon binding to Ca²⁺, effectively reducing background interference and improving detection sensitivity.
• Cell membrane permeability: The hydrophobic AM ester can passively cross the cell membrane. After being hydrolyzed by intracellular esterases, it forms a negatively charged fluorescent dye, enabling simple loading.
Intracellular calcium flux assays are essential tools for monitoring signal transduction pathways and conducting high-throughput screening of G protein-coupled receptors (GPCRs) and calcium channel targets. Since the introduction of Fluo-3 in 1989, successive generations of calcium indicators—including Fluo-4, Fluo-8®, and Cal-520®—have delivered progressively improved signal-to-background ratios, establishing themselves as standard indicators for confocal microscopy, flow cytometry, and high-throughput screening applications. Despite its widespread adoption, Fluo-4 presents significant limitations. Most notably, intracellular calcium assays using Fluo-4 AM require probenecid to prevent the dye from leaking out of cells—a requirement shared with its predecessor, Fluo-3. This dependency on probenecid compromises assay reliability, as probenecid exhibits well-documented, complex cellular effects that can confound results.
Calbryte 520 AM represents a next-generation solution to these challenges. This novel, cell-permeable calcium indicator enters cells in a non-fluorescent, inactive form. Once inside, intracellular esterases cleave the AM ester, activating the indicator and rendering it calcium-responsive. The hydrolyzed product becomes a polar molecule that cannot cross cell membranes, effectively trapping it within the cell—eliminating the need for probenecid. When bound to calcium ions, Calbryte 520 generates an exceptionally bright fluorescent signal with a superior signal-to-background ratio. Importantly, Calbryte 520 shares the same excitation and emission wavelengths as Fluo-4, allowing seamless integration into existing Fluo-4 assay protocols. The combination of enhanced signal quality, improved intracellular retention, and probenecid-free operation makes Calbryte 520 AM the most robust calcium indicator available for evaluating GPCR and calcium channel targets, as well as screening their agonists and antagonists in living cells.
Experimental Protocol
Preparation of stock solutions
Prepare a 2 to 5 mM stock solution of Cal-520 AM in high-quality, anhydrous DMSO. Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Calbryte 520 AM Stock Solution
1. Prepare a 2 to 5 mM stock solution of Calbryte 520 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte 520 AM is a clear, colorless solution.
Preparation of working solution
Calbryte 520 AM Working Solution
1. On the day of the experiment, either dissolve Calbryte 520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
2. Prepare a 2 to 20 µM Calbryte 520 AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Calbryte 520 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Calbryte 520 AM.
Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators.
Sample experimental protocol
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
1. Prepare cells in growth medium overnight.
2. On the next day, add 1X Calbryte 520 AM working solution to your cell plate. Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note: Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
4. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
5. Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
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