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即用型免洗Fluo-4钙离子实时检测试剂盒
有货
为什么选择此级别
BioReagent,无菌,无菌过滤,用于显微镜,适用于荧光分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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避光,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
即用型免洗Fluo-4钙离子实时检测试剂盒是精准检测细胞内钙离子浓度及动态变化的专业工具，为细胞生物学研究、疾病机制探索及药物研发提供关键技术支持。
钙是哺乳动物体内含量最丰富的矿物质，同时也是调控众多细胞生命活动的关键内源性因子。它主要以两种形式存在：自由离子形式与结合钙离子复合物形式 (如构成骨骼组织的磷酸钙、碳酸钙复合物)。从肌肉收缩、细胞粘附、激素/神经递质释放，到糖原代谢、细胞增殖/分化、血凝及神经突触传递，再到骨骼结构支持，大量生理过程均依赖钙信号的精准调控。一旦细胞特异性钙信号系统的完整性受损，便可能诱发多种疾病，因此精准检测细胞内钙离子浓度及动态变化具有重要科研与应用价值。
本试剂盒以Fluo-4 AM钙离子荧光探针为核心检测成分 (Fluo-4 AM是在Fluo-3 AM基础上升级的钙离子浓度指示剂)，其检测原理如下：Fluo-4 AM本身无荧光特性，进入细胞后会被胞内酯酶水解为Fluo-4；Fluo-4 可与细胞内钙离子特异性结合，结合后会发出绿色荧光。当细胞外钙离子内流、细胞内储存钙离子释放，或细胞内钙离子外流/储存时，会导致细胞质内钙离子浓度升高或降低，进而引起绿色荧光强度相应增强或减弱，通过监测荧光强度变化，即可实现对细胞内钙离子浓度动态变化的实时检测。
此外，该试剂盒适配荧光显微镜、荧光酶标仪、流式细胞仪等多种检测平台，具有荧光信号更强、检测灵敏度更高的优势，可同时满足细胞内钙离子浓度定量检测与动态变化监测的需求。
注：效能等同Thermo Fisher Fluo-4 Calcium Imaging Kit。
应用范围：钙离子检测
产品特点：
1、10 min快速染色：试剂混匀后直接加样，孵育后无需洗涤细胞，大幅缩短实验时间与操作步骤；
2、仪器友好：无需切换双激发光及计算信号比值，单波长检测，降低操作门槛，仪器适配性强；
3、荧光强度更高：Fluo-4 AM与钙离子结合后荧光会增加60至80倍，荧光强度较Fluo-3 AM提升100%；
4、超高灵敏度：低背景信号干扰，提升检测灵敏度，轻松捕捉低幅度的钙信号波动；
5、细胞毒性低：细胞毒性显著低于Fluo-3 AM，染色后细胞活率＞90%，适合长期动态追踪；
6、亲和力适配性好：轻松捕捉低幅度的钙信号波动，低背景信号干扰，信号清晰无干扰，实验结果稳定可靠；
产品参数：
1、Ex/Em：494/516 nm；
2、光谱图：
产品组分：
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注：按照96孔板每孔0.2 µL Fluo-4 AM的体系计算。
注：本试剂盒配套组分C (检测缓冲液)为我司自主创新的染色缓冲液，配方经多重优化，可在检测期间持续维持细胞生理状态并提供基础营养，并显著提升染料稳定性。若样本类型差异导致用量不足，可直接用PBS、HBSS等常规缓冲液补足体积重悬，不影响检测结果。
注意事项：
1、Fluo-4 AM遇水极易分解，如果不能一次用完，建议将储液小量分装保存。
2、AM酯基易吸潮，故请确保从冰箱拿出后恢复至室温再开封。
3、Fluo-4 AM微量不易观察，恢复室温后瞬时离心确保粉末离心至管底。
4、荧光染料均存在淬灭问题，请尽量注意避光。
5、本产品仅限于科研用途并且不得存放于普通住宅内。
6、为了您的安全和健康，请遵循您所在常规实验室安全规定。
使用说明：
一、 实验前准备
1. 工作液制备：
(1) 将试剂盒取出，恢复室温备用。
(2) 以96孔板为例，每孔100 µL Fluo-4染色工作液的体系，先加入Fluo-4 AM和溶解度增强剂，充分混匀后加入组分C (检测缓冲液)。Fluo-4染色工作液配制见表1。（其它规格孔板加染色工作液量如下，48孔板加150 μL/孔，24孔板加250 μL/孔，12孔板加500 μL/孔，6孔板加1 mL/孔。）
表 1.Fluo-4染色工作液配制
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注：配制好的Fluo-4染色工作液现用现配、避光配制，不能冻存。Fluo-4染色工作液中Fluo-4 AM的浓度可以根据染色效果在0.2-2 µL之间适当调整。
(3) (可选)如果遇到细胞，如CHO，出现去酯化的荧光探针Fluo-4明显的外排现象时，建议添加染色增强剂组分D (染色增强剂)。使用时将组分D (染色增强剂)按照1: 100稀释到染色工作液中，配制成含1×染色增强剂的染色工作液。
注：细胞在含1×染色增强剂的溶液中孵育的时间不能超过2 h。
2. 仪器准备：
(1) 荧光显微镜：激发/发射波长Ex/Em：494/516 nm；
(2) 流式细胞仪：检测通道激发/发射波长Ex/Em：494/516 nm；
(3) 荧光酶标仪：激发/发射波长Ex/Em：494/516 nm。
3. 对照组设置：
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阴性对照组：对细胞不进行任何处理，为实验组处理后钙信号变化提供参考。
实验组：验证探针活性与细胞加载效率。
二、 操作步骤-以96孔板为例
方案一：荧光显微镜检测 (适用于悬浮细胞)
1. 细胞收集与洗涤：
(1) 收集待测悬浮细胞于离心管中，1000 rpm室温离心5 min，小心吸弃上清。
(2) 用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤细胞1次。
注：通常洗涤能更好地降低背景荧光，吸除培养液、检测缓冲液和PBS、HBSS等常规缓冲液时，最好使用真空泵，酚红或血清对于本试剂盒的检测有一定的干扰。
2. 细胞重悬与计数：
(1) 使用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液重悬细胞沉淀，进行细胞计数。
(2) 取 5×10⁴-10⁵个细胞，1000 rpm低速离心5 min，去除组分C (检测缓冲液)或PBS、HBSS等常规缓冲液。
注：显微镜法对细胞密度要求不严格，只要实验组与对照组密度一致即可。具体可根据样本种类与实验条件灵活调整，以显微镜下视野中细胞分布约70%-85%为宜。
3. 细胞染色：
(1) 参照表1配制Fluo-4染色工作液。
(2) 向96孔板中加入100 µL的Fluo-4染色工作液，37 ℃避光孵育30 min。孵育时间可在10-60 min进行调整，最快10min快速染色，最大程度保持细胞活性，简化实验流程。
注：如果首次实验不能确定孵育温度和时间，建议先37 ℃孵育30 min，观察荧光效果。如果细胞死亡较多，适当缩短时间；如果荧光强度太弱，适当延长时间。
注：本试剂盒为免洗涤型检测体系，无需洗涤即可进行荧光检测，孵育后也可加入组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤1-3次。
4. 显微镜观察与拍照：
将细胞悬液加入96孔板中，静置片刻，待细胞自然沉降贴底后，置于荧光显微镜下观察。
注：细胞悬液使用量可根据细胞计数的数量自行调整，显微镜视野中细胞分布70%-85%为最佳。
方案二：荧光显微镜检测 (适用于贴壁细胞)
1. 细胞处理：
提前一天在96孔板中接种细胞，使得细胞汇合度达到70%-85%，待细胞贴壁后按照实验设计处理细胞。
2. 细胞清洗：
(1) 小心吸弃细胞培养液。
(2) 加入100 μL的组分C (检测缓冲液)或PBS、HBSS等常规缓冲液轻柔洗涤细胞1次，洗涤后吸净组分C (检测缓冲液)或PBS、HBSS等常规缓冲液。
3. 细胞染色：
(1) 参照表1配制Fluo-4染色工作液。
(2) 将100 μL Fluo-4染色工作液加入细胞中，37 ℃避光孵育30 min。孵育时间可在10-60 min进行调整，最快10 min快速染色，最大程度保持细胞活性，简化实验流程。
注：如果首次实验不能确定孵育温度和时间，建议先37 ℃孵育30 min，观察荧光效果。如果细胞死亡较多，适当缩短时间；如果荧光强度太弱，适当延长时间。
注：本试剂盒为免洗涤型检测体系，无需洗涤即可进行荧光检测，孵育后也可加入组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤1-3次。
4. 显微镜观察与拍照：
(1) 洗涤后，每孔加入对应体积的组分C (检测缓冲液)或PBS、HBSS等常规缓冲液，以保持细胞湿润。
(2) 直接将培养板置于荧光显微镜下观察和拍照。滤光片设置同方案一。
方案三：流式细胞仪检测 (适用于悬浮细胞与贴壁细胞)
1. 细胞准备：
(1) 悬浮细胞：同方案一，取5×10⁴-10⁵ 个细胞。
(2) 贴壁细胞：
a. 吸弃培养液，用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液轻柔清洗1次。
b. 加入适量胰酶消化液 (覆盖细胞即可)，室温下置于显微镜下观察，待细胞变圆、间隙增大时，用移液器轻轻吹打，使细胞完全脱落。
c. 关键：立即加入含血清的完全培养基以终止消化。
d. 将细胞悬液转移至离心管，1000 rpm，室温离心5 min，吸弃上清。
e. 用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液重悬细胞并进行细胞计数。
f. 取5×10⁴-10⁵个细胞，室温1000 rpm低速离心5 min，吸弃上清。
2. 细胞染色：
(1) 参照表1配制Fluo-4染色工作液。
(2) 将1 mL的Fluo-4染色工作液加入细胞中，37 ℃避光孵育30 min。孵育时间可在10-60 min进行调整，最快10 min快速染色，最大程度保持细胞活性，简化实验流程。
注：如果首次实验不能确定孵育温度和时间，建议先37 ℃孵育30 min，观察荧光效果。如果细胞死亡较多，适当缩短时间；如果荧光强度太弱，适当延长时间。
注：本试剂盒为免洗涤型检测体系，无需洗涤即可进行荧光检测，孵育后也可加入组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤1-3次。
3. 上机检测：
(1) 重要：染色完成后1 h内进行流式检测，以保证荧光信号稳定。
(2) 用流式细胞仪在预设的检测通道下进行分析。
(3) 首先通过空白对照（未染色细胞）调节电压，使细胞群位于坐标系左下角。
(4) 其次检测对照组和实验组，确认实验体系成立。
(5) 最后检测实验样品，记录并分析细胞的平均荧光强度。
注：所有样本染色后需避光、置冰上并立即检测；定量实验 (如流式)尤须严格在1 h内完成，以确保数据准确、可重复。
方案四：荧光酶标仪检测 (适用于悬浮与贴壁细胞)
1. 接种培养：将细胞接种于96孔板黑色多孔板中，每孔的细胞数需要控制在100-10000个，通常宜在2000-5000个范围内。
2. 洗涤：收集待测悬浮细胞悬液至离心管中。
(1) 悬浮细胞：250-1000× g室温离心5 min，吸除上清，用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤1次。
(2) 贴壁细胞：吸除培养液，用组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤细胞1次。
注：通常洗涤能更好地降低背景荧光，吸除培养液和组分C (检测缓冲液)或 PBS、HBSS等常规缓冲液时最好使用真空泵，酚红或血清对于本试剂盒的检测有一定的干扰。
3. 细胞染色：
(1) 参照表1.配制Fluo-4染色工作液。
(2) 加入100 μL的Fluo-4染色工作液，37 ℃避光孵育30 min。孵育时间可在10-60 min进行调整，最快10 min快速染色，最大程度保持细胞活性，简化实验流程。
注：如果首次实验不能确定孵育温度和时间，建议先 37 ℃孵育 30 min，观察荧光效果。如果细胞死亡较多，适当缩短时间；如果荧光强度太弱，适当延长时间。
注：本试剂盒为免洗涤型检测体系，无需洗涤即可进行荧光检测，孵育后也可加入组分C (检测缓冲液)或PBS、HBSS等常规缓冲液洗涤1-3次。
4. 荧光酶标仪检测：
在荧光酶标仪预设的条件检测，通过对比对照组与处理组的RFU (Relative Fluorescence Units)，可得出药物刺激的效果。
三、 结果判读：
定性分析 (显微镜)：
图 1.Hela细胞钙离子浓度动态变化图
在荧光显微镜下：
图1为加入离子霉素的Hela细胞在同一视野下的动态变化，在荧光显微镜下观察细胞内绿色荧光的分布以及强度情况。当细胞受刺激而让钙离子浓度出现上升，那么Fluo-4标记细胞的荧光强度会明显变强，可实现对细胞内钙离子浓度的动态变化的实时监测。
常见疑问与解答：
1、问：为什么没有检测到荧光值或荧光值低？
答：可能是因为细胞密度不够，可增加细胞密度；同时也可以考虑延长孵育时间。
2、问：Fluo-4孵育后发现细胞死亡太多怎么办？
答：首先应评估染料的浓度和处理时间是否合适。Fluo-4 AM虽然相对较为安全，但过高的浓度或过长的染色时间仍可能导致细胞损伤。建议降低染料浓度或缩短染色时间，并在实验过程中密切观察细胞状态。
The Ready-to-Use Wash-Free Fluo-4 Calcium Ion Real-Time Detection Kit is a professional tool for the accurate measurement of intracellular calcium ion concentration and dynamic changes. It provides key technical support for cell biology research, disease mechanism investigation, and drug development.
Calcium is the most abundant mineral in mammals and a critical endogenous factor regulating numerous cellular activities. It exists mainly in two forms: free ionic calcium and calcium-binding complexes (e.g., calcium phosphate and calcium carbonate complexes in bone tissue). Many physiological processes rely on precise regulation of calcium signaling, including muscle contraction, cell adhesion, hormone/neurotransmitter release, glycogen metabolism, cell proliferation/differentiation, blood coagulation, synaptic transmission, and structural support of bone. Impairment of the integrity of cell-specific calcium signaling systems may induce multiple diseases. Therefore, accurate detection of intracellular calcium concentration and dynamics has important scientific and application value.
This kit uses Fluo-4 AM calcium fluorescent probe as the core component (an improved calcium indicator based on Fluo-3 AM). Its detection principle is as follows:Fluo-4 AM is non-fluorescent by itself. After entering cells, it is hydrolyzed by intracellular esterases into Fluo-4, which specifically binds to intracellular calcium ions and emits strong green fluorescence upon binding.Influx of extracellular calcium, release of calcium from intracellular stores, or calcium efflux/sequestration causes increases or decreases in cytoplasmic calcium concentration, leading to corresponding changes in green fluorescence intensity. By monitoring fluorescence intensity, real-time detection of dynamic changes in intracellular calcium concentration is achieved.
In addition, this kit is compatible with fluorescence microscopes, fluorescence microplate readers, flow cytometers, and other platforms. It features stronger fluorescence signals and higher sensitivity, supporting both quantitative detection and dynamic monitoring of intracellular calcium.
Note: Performance equivalent to Thermo Fisher Fluo-4 Calcium Imaging Kit.
Application
Calcium ion detection
Product Features
1. 10 min rapid staining: Direct loading after reagent mixing; no cell washing required after incubation, greatly reducing time and steps.
2. Instrument-friendly: Single-wavelength detection; no dual-excitation switching or ratio calculation required, lowering operational difficulty.
3. Higher fluorescence intensity: Fluorescence increases 60–80-fold upon calcium binding; fluorescence intensity is 100% higher than Fluo-3 AM.
4. Ultra-high sensitivity: Low background interference enables detection of small calcium signal fluctuations.
5. Low cytotoxicity: Significantly lower toxicity than Fluo-3 AM; cell viability >90% after staining, suitable for long-term dynamic tracking.
6. Good affinity & stability: Clearly captures weak calcium signals with low background, ensuring stable and reliable results.
Product Parameters
1. Ex/Em: 494/516 nm
2. Spectrum:
Precautions
1. Fluo-4 AM is highly unstable in water. Aliquot into small volumes if not used immediately.
2. AM esters are hygroscopic. Allow to warm to room temperature before opening.
3. Fluo-4 AM is difficult to visualize; briefly centrifuge to collect powder at the bottom after equilibration.
4. All fluorescent dyes are prone to photobleaching; protect from light.
5. This product is for research use only and must not be stored in residential areas.
6. Follow general laboratory safety guidelines for your health and safety.
Instructions for Use
I. Pre-Experiment Preparation
1. Working Solution Preparation
(1) Remove the kit and equilibrate to room temperature.
(2) Taking a 96‑well plate as an example, for a 100 µL Fluo‑4 Staining Working Solution system per well, first add Fluo‑4 AM and Solubility Enhancer, mix thoroughly, then add Component C (Assay Buffer). (For other plate formats, the volume of staining working solution per well is as follows: 150 μL/well for 48‑well plates, 250 μL/well for 24‑well plates, 500 μL/well for 12‑well plates, 1 mL/well for 6‑well plates.)
Table 1. Fluo-4 Staining Solution Preparation
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Note: The prepared Fluo-4 Staining Working Solution must be prepared freshly and used immediately, prepared under light protection, and cannot be frozen. The volume of Fluo-4 AM in the working solution can be appropriately adjusted between 0.2-2 µL according to staining performance.
(3) (Optional) If obvious efflux of de-esterified Fluo-4 fluorescent probe occurs in certain cell lines such as CHO cells, it is recommended to add Component D (Staining Enhancer). During use, dilute Component D at a ratio of 1:100 into the staining working solution to prepare staining working solution containing 1× Staining Enhancer.
Note: The incubation time of cells in solution containing 1× Staining Enhancer must not exceed 2 h.
2. Instrument Preparation
(1) Fluorescence microscope: Ex/Em = 494/516 nm
(2) Flow cytometer: Detection channel Ex/Em = 494/516 nm
(3) Fluorescence microplate reader: Ex/Em = 494/516 nm
3. Control Group Setup
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Negative Control: Cells without any treatment, used as a reference for calcium signal changes in the test group.
Test Group: Used to verify probe activity and cellular loading efficiency.
II. Procedure (Taking 96‑well plate as an example)
Protocol A: Fluorescence Microscopy (For suspension cells)
1. Cell Collection and Washing
(1) Collect suspension cells into a centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, and carefully aspirate the supernatant.
(2) Wash cells once with Component C (Assay Buffer), PBS, HBSS or other conventional buffers.
Note: Washing generally reduces background fluorescence. Aspiration of medium, Assay Buffer, PBS, HBSS, etc., is best performed with a vacuum pump. Phenol red or serum may interfere with detection using this kit.
2. Cell Resuspension and Counting
(1) Resuspend the cell pellet with Component C (Assay Buffer), PBS, HBSS or other conventional buffers, and perform cell counting.
(2) Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min at low speed, and remove the buffer.
Note: The microscopy method is not strict on cell density, as long as the density is consistent between test and control groups. Adjust according to cell type and experimental conditions; optimal cell distribution under the microscope is 70-85%.
3. Cell Staining
(1) Prepare Fluo-4 Staining Working Solution according to Table 1.
(2) Add 100 µL of Fluo-4 Staining Working Solution to each well of a 96‑well plate, incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted from 10-60 min; rapid staining in 10 min preserves cell viability and simplifies the procedure.
Note: If incubation temperature and time are uncertain for the first experiment, incubate at 37 ℃ for 30 min and observe fluorescence. Shorten time if excessive cell death occurs; extend time if fluorescence intensity is too low.
Note: This kit is a wash-free detection system. No washing is required before detection. Optional washing 1-3 times with Component C, PBS or HBSS is allowed after incubation.
4. Microscopic Observation and Imaging
Add the cell suspension to the 96‑well plate, let stand briefly until cells settle naturally, then observe under a fluorescence microscope.
Note: The volume of cell suspension can be adjusted according to cell count. Optimal cell distribution in the microscope field is 70-85%.
Protocol B: Fluorescence Microscopy (For adherent cells)
1. Cell Treatment
Seed cells in a 96‑well plate one day in advance to reach 70–85% confluency. After cells adhere, treat them according to the experimental design.
2. Cell Washing
(1) Carefully aspirate the cell culture medium.(2) Add 100 μL of Component C (Assay Buffer), PBS, HBSS or other conventional buffers to gently wash cells once, then completely aspirate the buffer.
3. Cell Staining
(1) Prepare Fluo-4 Staining Working Solution according to Table 1.
(2) Add 100 μL of Fluo-4 Staining Working Solution to the cells, incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted from 10-60 min.
Note: If conditions are uncertain, first incubate at 37 ℃ for 30 min. Adjust time according to cell viability and fluorescence intensity.
Note: This is a wash-free system; no washing is required. Optional washing 1-3 times is allowed.
4. Microscopic Observation and Imaging
(1) After washing, add an appropriate volume of Component C (Assay Buffer), PBS or HBSS to keep cells moist.
(2) Place the plate directly under the fluorescence microscope for observation and imaging. Filter settings are the same as Protocol A.
Protocol C: Flow Cytometry (For suspension and adherent cells)
1. Cell Preparation
(1) Suspension cells: Same as Protocol A; take 5×10⁴–1×10⁵ cells.
(2) Adherent cells:
a. Aspirate medium, gently wash once with Component C (Assay Buffer), PBS or HBSS.
b. Add an appropriate amount of trypsin (just covering the cells), observe under a microscope at room temperature. When cells round up and gaps increase, gently pipette to detach cells completely.
c. Critical: Immediately add complete medium containing serum to terminate digestion.
d. Transfer cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, aspirate supernatant.
e. Resuspend cells with buffer and count.
f. Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 min, aspirate supernatant.
2. Cell Staining
(1) Prepare Fluo-4 Staining Working Solution according to Table 1.
(2) Add 1 mL of staining solution, incubate at 37 ℃ for 30 min in the dark. Time can be adjusted from 10-60 min.
Note: Adjust incubation time based on cell viability and fluorescence intensity.Note: Wash-free system; no washing required. Optional washing 1-3 times is allowed.
3. Detection
(1) Important: Perform flow cytometry detection within 1 h after staining to maintain stable fluorescence signals.
(2) Analyze using a flow cytometer with preset detection channels.
(3) First, adjust voltage using unstained cells (blank control) so that the cell population is located in the lower-left corner of the dot plot.
(4) Then detect control and test groups to validate the system.
(5) Finally detect samples, record and analyze mean fluorescence intensity (MFI).
Note: All stained samples must be kept on ice, protected from light, and detected immediately. Quantitative assays (e.g., flow cytometry) must be completed within 1 h to ensure accurate and reproducible results.
Protocol D: Fluorescence Microplate Reader (For suspension and adherent cells)
1. Seeding and Culture
Seed cells in a black 96‑well plate. Cell number per well: 100-10000, recommended 2000-5000.
2. Washing
(1) Suspension cells: Centrifuge at 250-1000× g for 5 min at room temperature, aspirate supernatant, wash once with Component C (Assay Buffer), PBS or HBSS.
(2) Adherent cells: Aspirate medium, wash once with Component C (Assay Buffer), PBS or HBSS.
Note: Washing reduces background fluorescence. Use a vacuum pump for aspiration. Phenol red or serum may interfere with detection.
3. Cell Staining
(1) Prepare Fluo-4 Staining Working Solution according to Table 1.
(2) Add 100 μL per well, incubate at 37 ℃ for 30 min in the dark. Time can be adjusted from 10-60 min.
Note: Adjust incubation time based on cell viability and fluorescence intensity.
Note: Wash-free system; no washing required. Optional washing 1-3 times is allowed.
4. Detection with Fluorescence Microplate Reader
Detect under preset conditions. The effect of drug stimulation can be determined by comparing RFU (Relative Fluorescence Units) between control and treated groups.
III. Result Interpretation
Qualitative Analysis (Microscopy)
Figure 1. Dynamic changes of intracellular calcium concentration in Hela cells
Under fluorescence microscopy:
Figure 1 shows dynamic changes in Hela cells stimulated with ionomycin in the same field. The distribution and intensity of green fluorescence in cells are observed.When cells are stimulated and intracellular calcium concentration increases, the fluorescence intensity of Fluo-4 labeled cells is significantly enhanced, allowing real‑time monitoring of dynamic changes in intracellular calcium concentration.
Frequently Asked Questions
1. Q: No fluorescence signal or low fluorescence value detected?
A: Possible low cell density; increase cell number. Alternatively, extend the incubation time.
2. Q: Excessive cell death after Fluo-4 incubation?
A: First evaluate dye concentration and incubation time. Although Fluo-4 AM is relatively safe, excessively high concentration or prolonged staining may cause cell damage. It is recommended to reduce dye concentration or shorten staining time, and closely monitor cell status during the experiment.
规格
别名
Fluo-4钙离子实时检测试剂盒
英文别名
Fluo-4 Calcium Real-Time Detection Kit
规格或纯度
无菌过滤,BioReagent,用于显微镜,无菌,适用于荧光分析
英文名称
Ready-to-Use Wash-Free Fluo-4 Calcium Ion Real-Time Detection Kit
应用
Fluorescence Analysis, 细胞染色, 钙离子检测
敏感性
Light-sensitive
Emission(nm)
516nm
Excitation(nm)
Blue Laser(488nm)
Ex/Em(nm)
Ex:494nm;Em:516nm
储存与运输
储存条件
避光,-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (36 months). Store in the dark.
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