钙离子测量对众多生物学研究至关重要。能对钙结合产生光谱响应的荧光探针使研究人员得以通过荧光显微镜、流式细胞仪、荧光光谱仪和荧光酶标仪等技术检测细胞内游离钙离子浓度的变化。在红色荧光钙指示剂中，Rhod-2是目前最常用的探针。但Rhod-2 AM酯酶水解后在活细胞中的荧光强度较弱，且钙响应信号变化微弱。
Cal-590的开发正是为了在保持与Rhod-2相同光谱波长（可兼容TRITC/Cy3®滤光片系统）的同时，显著改善细胞加载效率和钙响应灵敏度。在CHO和HEK细胞中，Cal-590 AM的钙响应灵敏度远超Rhod-2 AM。其发射光谱与FITC、Alexa Fluor® 488及GFP能实现良好区分，因此成为GFP细胞系或FITC/Alexa Fluor® 488标记抗体多重细胞内检测的理想钙探针。
产品特点
1.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。
2.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。
光谱性质
消光系数 (cm⁻¹・M⁻¹)：78000
激发波长 (nm)：574
发射波长 (nm)：588
荧光量子产率：0.62
适用仪器
| 荧光显微镜 | |
| Ex: | TRITC/Cy3 滤波片组 |
| Em： | TRITC/Cy3 滤波片组 |
| 推荐孔板: | 黑色透明底板 |
| 荧光酶标仪 | |
| Ex: | 540 nm |
| Em： | 590 nm |
| Cutoff： | 570 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。
Cal-590® AM 储备溶液
使用高质量无水DMSO配制2-5 mM的Cal-590® AM储备液。
注意：Cal-590® AM溶于DMSO后为无色透明溶液。
工作溶液配制
Cal-590® AM 工作溶液
实验当天，将Cal-590® AM溶解于DMSO或将储备液 aliquot 解冻至室温。
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-590® 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-590® 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-590® 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TRITC/CY3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 = 540/590 nm cutoff 570 nm检测荧光信号
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-2 is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Cal-590 has been developed to improve Rhod-2 cell loading and calcium response while maintaining the spectral wavelength of Rhod-2, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells Cal-590 AM has cellular calcium response that is much more sensitive than Rhod-2 AM. The spectra of Cal-590 is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.
Product Features
1. Low background fluorescence: Fluorescence intensity increases significantly upon binding to Ca²⁺, effectively reducing background interference and improving detection sensitivity.
2. Cell membrane permeability: The hydrophobic AM ester can passively cross the cell membrane. After hydrolysis by intracellular esterases, it is converted into negatively charged fluorescent dye, enabling simple cell loading.
Spectral properties
Extinction coefficient (cm⁻¹ M⁻¹):
| 78000 |
| Excitation (nm): | 574 |
| Emission (nm): | 588 |
| Quantum yield: | 0.62 |
Instrument settings| Fluorescence microscope | | Excitation: | TRITC/Cy3 | | Emission: | TRITC/Cy3 | | Recommended plate: | Black wall/clear bottom |
|
Fluorescence microplate reader | | Excitation: | 540 | | Emission: | 590 | | Cutoff: | 570 | | 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-590 AM Stock Solution
1. Prepare a 2 to 5 mM stock solution of Cal-590 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-590 AM is a clear, colorless solution.
Preparation of working solution
Cal-590 AM Working Solution
1. On the day of the experiment, either dissolve Cal-590 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
2. Prepare a 2 to 20 µM Cal-590 AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F127. For most cell lines, Cal-590 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-590 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 deesterified 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-590 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 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 TRITC/Cy3 filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 540/590 nm cutoff 570 nm.
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