Cal‑520 AM 是一种细胞渗透性钙离子指示剂，相比传统指示剂具有更高的亮度与更优的细胞滞留性，可为活细胞内钙离子动员提供可靠的荧光检测方案。
• 其信噪比显著提升
• 即便不使用丙磺舒，仍能实现优异的胞内滞留效果
• 可取代传统的钙离子指示剂（如 Fluo-3 与 Fluo-4）
产品介绍
作用机制与原理
在表达特定GPCR（G蛋白偶联受体）或钙离子通道的细胞中，钙信号传导可通过预载Cal-520 AM进行检测。Cal-520 AM可自由穿过细胞膜进入胞内。进入细胞后，其疏水性封闭基团会被酯酶水解，从而生成带负电荷的荧光染料并滞留于细胞内。该染料与钙离子结合后，荧光强度会大幅增强。当使用激动剂刺激细胞时，受体会引发胞内钙离子释放，从而显著增强Cal-520®的荧光信号。
应用
Cal-520 AM具有长波长、高灵敏度及超过100倍荧光增强等特性，使其成为检测细胞钙信号的理想指示剂。其高信噪比与优异的胞内滞留性，使Cal-520钙检测体系成为评估GPCR及钙通道靶点、筛选相应激动剂与拮抗剂的可靠工具。
光谱性质
激发波长 (nm)：492
发射波长 (nm)：515
荧光量子产率：0.75
适用仪器
荧光显微镜
激发： FITC
发射： FITC
推荐孔板： 黑色透明
荧光酶标仪
激发： 490nm
发射： 525nm
cutoff: 515nm
推荐孔板： 黑色透明
读取模式： 底读模式
实验方案
储备液配制
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。
Cal‑520 AM 储备液
使用高质量无水DMSO配制2-5 mM的Cal-520 AM储备液。
例如：
a.Cal-520 AM的用量：1mg
b.所需浓度：2 mM
c.在合适的容器中，将1mg的Cal-520 AM与453.33μL的无水DMSO混合。
注意：Cal-520 AM溶于DMSO后为无色透明溶液。
工作溶液配制
Cal‑520 AM 工作液
1. 实验当天，将Cal-520 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Cal-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Cal-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 Cal-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。
注意：在部分细胞系中，染料孵育超过 2 小时可提升信号强度。
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
附加信息
1 M NaOH配方：
1.在合适的容器中准备2 mL蒸馏水。
2.在混合下向溶液中缓慢加入100mg NaOH。
3.加入蒸馏水至体积为2.5 mL。
4.在室温下将溶液储存在塑料容器中。
注意：步骤2这是一个放热过程，应遵循适当的预防措施和指南。
10％Pluronic F-127配方：
1.将1g Pluronic®F-127（Cat#20050）溶解在10毫升蒸馏水中，制成10％（w / v）储备溶液。
2.在40至50℃的温度下加热10％Pluronic F-127储备溶液约30分钟。
3.根据其储存规格储存多余的10％Pluronic®F-127。
25 mM Probenecid配方：
1.在合适的容器中，将1小瓶（72mg）的丙磺舒溶解在0.3mL的1M NaOH中。
2.加入HHBS或您选择的缓冲液，直至体积为10 mL。
3.根据其储存规格分装并储存任何未使用的25 mM 丙磺舒溶液。
Cal-520 AM is a cell-permeable calcium indicator with enhanced brightness and improved cellular retention compared to traditional indicators, providing robust fluorescence-based detection of intracellular calcium mobilization in live cells.
• Significantly improved signal to noise (S/N) ratio
• Excellent intracellular rentention, even without probenecid
• Replaces outdated calcium indicators such as Fluo-3 and Fluo-4
Product Description
Mechanism and Principle
Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520® AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal-520 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520.
Application
The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520 AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520 calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.
Spectral properties
| Excitation (nm): | 492 |
| Emission (nm): | 515 |
| Quantum yield: | 0.75 |
Instrument settings
| Fluorescence microscope |
| Excitation: | FITC |
| Emission: | FITC |
| Recommended plate: | Black wall/clear bottom |
|
| Fluorescence microplate reader |
| Excitation: | 490 |
| Emission: | 525 |
| Cutoff: | 515 |
| Recommended plate: | Black wall/clear bottom |
| Instrument specification(s): | Bottom read mode/Programmable liquid handling |
Experimental Protocol
Preparation of stock solutions
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.
Cal-520 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Cal-520 AM in high-quality, anhydrous DMSO.
For example:
a. Mass of Cal-520 AM: 1 mg
b. Target concentration: 2 mM
c. In a suitable container, mix 1 mg of Cal-520 AM with 453.33 μL of anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-520 AM is a clear, colorless solution.
Preparation of working solution
Cal-520 AM Working Solution
1. On the day of the experiment, either dissolve Cal-520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
2. Prepare a 2 to 20 μM Cal-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, Cal-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 Cal-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 Cal-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 ℃ for 1 to 2 hours.
Note: Incubating the dye for longer than 2 hours 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.
Additional Information
1 M NaOH Formula:
1. Prepare 2 mL of distilled water in a suitable container.
2. Slowly add 100 mg NaOH to the solution while mixing.
3. Add distilled water up to a final volume of 2.5 mL.
4. Store the solution in a plastic container at room temperature.
Note: Step 2 is an exothermic process; appropriate precautions and guidelines should be followed.
10% Pluronic F-127 Formula:
1. Dissolve 1 g Pluronic® F-127 (Cat#20050) in 10 mL distilled water to prepare a 10% (w/v) stock solution.
2. Heat the 10% Pluronic F-127 stock solution at 40–50 °C for approximately 30 minutes.
3. Store the remaining 10% Pluronic® F-127 according to its storage specifications.
25 mM Probenecid Formula:
1. In a suitable container, dissolve one vial (72 mg) of probenecid in 0.3 mL of 1 M NaOH.
2. Add HHBS or your chosen buffer to bring the final volume to 10 mL.
3. Aliquot and store any unused 25 mM probenecid solution according to its storage specifications.
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