钙离子检测在众多生物学研究中具有关键作用。能与Ca²⁺结合产生特征性光谱响应的荧光探针，使科研人员能够通过荧光显微镜、流式细胞仪、荧光分光光度计和荧光酶标仪等技术手段，精确监测活细胞内游离Ca²⁺浓度的动态变化。在可见光激发的钙离子指示剂中，Fluo-3 AM和Fluo-4 AM是活细胞钙成像最常用的探针，但其经酯酶水解后在活细胞中的荧光强度中等，且需要较苛刻的细胞加载条件才能获得最佳钙响应信号。
Fluo-8系列染料在保持与Fluo-3/Fluo-4相近光谱特性（激发/发射波长约490/520 nm）的同时，显著改善了细胞加载效率和钙响应性能。相较于Fluo-3 AM和Fluo-4 AM必须37°C加载的条件，Fluo-8, AM可在室温下完成细胞加载，其荧光强度达到Fluo-4 AM的2倍、Fluo-3 AM的4倍。
一、适用仪器
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 |
|
二、实验方案
储备溶液配制
所有未使用的储备液需分装为单次用量，于-20°C避光保存，避免反复冻融
Fluo-8, AM 储备液
用高质量无水 DMSO 制备 2-5 mM Fluo-8, AM 储备溶液。
工作溶液配制
Fluo-8, AM 工作溶液
1. 在实验当天，将Fluo-8, AM溶解在DMSO中，或将储备溶液的等分试样解冻至室温。
2. 用含0.04% Pluronic® F-127的缓冲液（如Hanks-Hepes）稀释至2-20 μM。大多数细胞系，建议使用终浓度为 4-5 μM 的 Fluo-8, AM。必须需根据细胞类型调整浓度
注意： 1.Pluronic® F-127 可增强 Fluo-8, AM 的水溶性。
2.若细胞存在有机阴离子转运体，建议在工作液中添加1-2 mM丙磺舒（孔内终浓度0.5-1 mM）以防止水解产物外排。
操作步骤
以下是推荐的将AM酯加载到活细胞中的方案。本方案仅提供指南，实际应根据您的具体需求进行修改。
1. 在生长培养基中制备细胞过夜。
2. 第二天，将 1×Fluo-8, AM 工作溶液添加到孔板中。
注意：如果您的化合物会干扰血清，请在加载染料之前用新鲜的 HHBS 缓冲液替换生长培养基。
3. 将加入染料的孔板在37°C的细胞培养箱中培养30至60分钟。
注意 将染料孵育 2 小时以上可以提高某些细胞系的信号强度。
4. 用 HHBS 或您选择的缓冲液（含有阴离子转运蛋白抑制剂，例如 1 mM 丙磺舒，如果适用）替换染料工作溶液，以去除多余的探针。
5. 根据需要添加刺激物，同时使用配备有 FITC 滤光片组的荧光显微镜或 FDSS、FLIPR 或 FlexStation的荧光板读取器在 490/525 nm 截止波长 515 nm 处检测荧光。
示例数据分析与图表
/
图 1. 将 U2OS 细胞以 40,000 个细胞 / 100 微升 / 孔的密度接种到 96 孔黑壁透明底康宁（Costar）培养板中，过夜培养。移除生长培养基后，分别向孔中加入 100 微升含 4 微摩（μM）Fluo-3 AM、Fluo-4 AM 和 Fluo-8 AM 的汉克斯 - 羟乙基哌嗪乙磺酸缓冲液（HHBS），在 37℃、5% 二氧化碳（CO₂）培养箱中孵育 1 小时。用 200 微升汉克斯 - 羟乙基哌嗪乙磺酸缓冲液（HHBS）洗涤细胞两次，随后使用奥林巴斯（Olympus）IX71 荧光显微镜，通过异硫氰酸荧光素（FITC）通道对细胞进行成像。
Calcium measurements are critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca²⁺ have enabled researchers to investigate changes in intracellular free Ca²⁺ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy, and fluorescence microplate readers. Fluo-3 AM and Fluo-4 AM are most commonly used among the visible light-excitable calcium indicators for live-cell calcium imaging. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8 dyes are developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of Ex/Em = ∼490/∼520 nm. Fluo-8, AM can be loaded into cells at room temperature, while Fluo-3 AM and Fluo-4 AM require 37°C for cell loading. In addition, Fluo-8, AM is two times brighter than Fluo-4 AM and four times brighter than Fluo-3 AM.
KEY PARAMETERS
Fluorescence microscope
| Emission | FITC | | Excitation | FITC | | Recommended plate | Black wall/clear bottom |
|
Fluorescence microplate reader
| Cutoff | 515 | | Emission | 525 | | Excitation | 490 | | Recommended plate | Black wall/clear bottom | | Instrument specification(s) | Bottom read mode/Programmable liquid handling |
|
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.
Fluo-8, AM Stock Solution
- Prepare a 2 to 5 mM stock solution of Fluo-8, AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
Fluo-8, AM Working Solution
1. On the day of the experiment, either dissolve Fluo-8, AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
2. Prepare a 2 to 20 µM Fluo-8, 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, Fluo-8, 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 Fluo-8, 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 1×Fluo-8, 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 FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at 490/525 nm (cutoff 515 nm).
EXAMPLE DATA ANALYSIS AND FIGURES
Figure 1. U2OS cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8, AM in HHBS at a concentration of 4 μM in a 37 °C, 5% CO₂ incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
ZJ26F0232350
危险品化学品经营许可证（带存储）