Fluo-8 AM Calcium Probe是精准检测细胞内钙离子浓度变化的专业工具，本产品以Fluo-8 AM钙离子荧光探针为核心检测成分，其检测原理如下：Fluo-8 AM是Fluo-8的一种乙酰甲酯衍生物，具有细胞膜渗透性，只需简单培养，即可轻易进入细胞，一旦进入细胞内即被其内酯酶剪切生成不具膜渗透性的Fluo-8，从而滞留在胞内以发挥相应生理功能。本产品荧光显微镜、荧光酶标仪、流式细胞仪等多种检测平台检测钙离子浓度变化，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。
Fluo-8 AM作为经典钙指示剂Fluo-4 AM、Fluo-3 AM的升级产品，在性能上实现全面优化。Fluo-3 AM和Fluo-4 AM是在活细胞钙成像研究里最为常用的可见光可激发钙指示剂，其在活细胞中仅能呈现出中等强度的荧光，而且为使细胞钙反应达到最佳效果，需要较为苛刻的细胞加载条件。Fluo-8 AM可在室温条件下进行细胞染色，Fluo-3 AM和Fluo-4 AM则必须在 37 ℃的环境中才能进行细胞上样操作。Fluo-8 AM在显著改善了细胞负载情况以及钙反应效果。Fluo-8 AM在荧光亮度上是Fluo-4 AM的2倍，是Fluo-3 AM的4倍，完美替代Fluo-3 AM和Fluo-4 AM。
注：效能等同AAT Fluo-8®, AM；
注：本文中Fluo-8®为AAT Bioquest的商标及注册商标。
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，孵育后最快10 min染色，大幅缩短实验时间与操作步骤；
2、加载条件灵活：室温或 37 °C孵育均可实现良好染色效果，无需较为苛刻的细胞加载条件；
3、荧光强度更高：荧光亮度为Fluo-4 AM的2倍、Fluo-3 AM的4倍，显著提升检测信号辨识度；
4、超高灵敏度：低背景信号干扰，检测灵敏度高，轻松捕捉低幅度的钙信号波动。
产品参数：
1、外观：可溶于DMSO的固体粉末；
2、CAS 号：1345980-40-6；
3、Ex/Em：494/516 nm；
4、分子式：C₅₀H₅₀N₂O₂₃；
5、分子量：1046.93；
6、光谱图：
产品组分：
| 组分 | 2300 T | | Fluo-8 AM Calcium Probe | 1 mg |
|
注：以每次使用100 μL染色工作液，染色工作液浓度4 μM计算。
注意事项：
1、AM酯基易吸潮，故请确保从冰箱拿出后恢复至室温再开封，Fluo-8 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。
2、Fluo-8 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。
3、荧光染料均存在淬灭问题，请尽量注意避光。
4、本产品仅限于科研用途并且不得存放于普通住宅内。
5、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. 储液准备：
将Fluo-8 AM从冰箱取出，恢复至室温后，瞬时离心至管底。用无水DMSO制备2-5 mM的Fluo-8 AM储备液。举例：制备5 mM储液需要往1 mg管子中加入191 µL无水DMSO。
注：配置好的储液建议分装保存放置-20 ℃，且最好用封口膜缠紧管盖。
2. 工作液准备：
(1) 用PBS或HBSS稀释Fluo-8 AM储液制备4 μM的Fluo-8 AM工作液。对于大多数细胞系，建议使用终浓度为4-5 μM的Fluo-8 AM工作液。
注：不同细胞所需上样浓度不一致，建议正式实验前做最佳浓度摸索。为了避免过度加载造成细胞毒性，建议在取得有效结果的基础上使用最低探针浓度，可从4 μM开始摸索。
(2) (可选)如果Fluo-8 AM进入细胞的效果不好，可向Fluo-8 AM溶液中加入适量20% Pluronic F-127溶液，防止Fluo-8AM在缓冲液中聚集并促进Fluo-8 AM进入细胞，Pluronic F-127终浓度控制在0.04~0.05%。
注：(1) 配制 20% (w/v)的Pluronic F-127 DMSO母液：100 mg Pluronic F-127中加入0.5 mL DMSO，配制成20% (w/v)的DMSO母液。溶解需要在40-50 ℃加热20-30 min。溶解后室温保存，勿冷藏。如果有结晶析出，可以重新加热后溶解，不影响使用；
(2) 使用Pluronic F-127 可降低Fluo-8 AM的稳定性，因此只建议在配制工作液时加入，不建议将其加入储液中；
(3) 如果您的细胞含有有机阴离子转运蛋白，则可以将丙磺舒 (1-2 mM)添加到染料工作溶液中 (最终浓度为0.5-1 mM)，以减少脱酯指示剂的泄漏。
3. 仪器准备：
(1) 荧光显微镜，Ex/Em：494/516 nm；
(2) 流式细胞仪，Ex/Em：494/516 nm；
(3) 荧光酶标仪，Ex/Em：494/516 nm。
4. 对照组设置：
| 组别 | Fluo-8 AM Calcium Probe | 样品细胞 | | 阴性对照 | + | 不做任何处理 | | 实验组 | + | 经实验处理细胞 |
|
(1) 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。
(2) 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PBS或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、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PBS或HBSS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PBS或HBSS。
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) 96孔板中加入100 µL的Fluo-8 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。
(2) 孵育后可加入PBS或HBSS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PBS或HBSS轻柔洗涤细胞1次，洗涤后吸净PBS或HBSS。
3. 细胞染色：
(1) 96孔板中加入100 µL的Fluo-8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。
(2) 孵育后可加入PBS或HBSS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PBS或HBSS，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。
(2) 贴壁细胞：
a. 吸弃培养液，用PBS或HBSS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PBS或HBSS重悬细胞并进行细胞计数。
f. 取10⁶个细胞，室温1000 rpm低速离心5 min，吸弃上清。
2. 细胞染色：
(1) 将1 mL的Fluo-8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。
(2) 孵育后可加入PBS或HBSS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PBS或HBSS常规缓冲液洗涤1次。
(2) 贴壁细胞：吸除培养液，用PBS或HBSS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PBS或HBSS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) 96孔板中加入100 µL的Fluo-8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。
(2) 孵育后可加入PBS或HBSS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：
1、定性分析 (显微镜)：
图1. SH-SY5Y细胞钙离子浓度动态变化图
在荧光显微镜下：
图1为对比不同处理组在Fluo (钙信号)和DCFDA (ROS 信号)在荧光显微镜下观察下的荧光强度情况，可系统分析不同刺激因素对细胞钙信号通路和氧化应激的协同或独立影响。
注：图片引自《Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release》(doi:10.1038/s41598-023-38210-w.)
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-8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-8 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。
Fluo-8 AM Calcium Probe is a professional tool for the accurate detection of intracellular calcium ion concentration changes. Centered on Fluo-8 AM calcium ion fluorescent probe as the core detection component, its detection principle is as follows: Fluo-8 AM is an acetoxymethyl ester derivative of Fluo-8 with cell membrane permeability. It can easily enter cells through simple incubation. Once inside the cell, it is cleaved by intracellular esterases to form Fluo-8, which lacks membrane permeability and thus accumulates in the cytoplasm to exert its corresponding physiological functions. This product is compatible with multiple detection platforms such as fluorescence microscopes, fluorescent microplate readers, and flow cytometers for measuring calcium ion concentration changes, providing critical technical support for cell biology research, disease mechanism exploration, and drug development.
As an upgraded product of the classic calcium indicators Fluo-4 AM and Fluo-3 AM, Fluo-8 AM achieves comprehensive performance optimization. Fluo-3 AM and Fluo-4 AM are the most commonly used visible-light-excitable calcium indicators in live-cell calcium imaging studies, but they only exhibit moderate fluorescence intensity in live cells and require relatively stringent cell loading conditions to achieve optimal calcium responses. In contrast, Fluo-8 AM allows cell staining at room temperature, whereas Fluo-3 AM and Fluo-4 AM must be loaded at 37 °C. Fluo-8 AM significantly improves cell loading efficiency and calcium response performance. Its fluorescence brightness is twice that of Fluo-4 AM and four times that of Fluo-3 AM, making it an ideal replacement for both.
Note: Equivalent in efficacy to AAT Fluo-8®, AM.
Note: Fluo-8® in this document is a trademark and registered trademark of AAT Bioquest.
Application
Calcium Ion Detection
Product Features
1. 10-Minute Rapid Staining: Add the mixed reagent directly to samples; staining can be completed in as little as 10 minutes after incubation, greatly reducing experimental time and operational steps.
2. Flexible Loading Conditions: Achieves excellent staining at either room temperature or 37 °C incubation, eliminating the need for stringent cell loading conditions.
3. Higher Fluorescence Intensity: Fluorescence brightness is twice that of Fluo-4 AM and four times that of Fluo-3 AM, significantly enhancing the distinguishability of detection signals.
4. Ultra-High Sensitivity: Low background signal interference and high detection sensitivity enable easy capture of low-amplitude calcium signal fluctuations.
Product Specifications
1. Appearance: Solid powder soluble in DMSO
2. CAS No.: 1345980-40-6
3. Ex/Em: 494/516 nm
4. Molecular Formula: C₅₀H₅₀N₂O₂₃
5. Molecular Weight: 1046.93
6. Spectrum:
Product Components
Component
| 2300 T | | Fluo-8 AM Calcium Probe | 1 mg |
|
Note: Calculated based on using 100 μL of staining working solution at a concentration of 4 μM per assay.
Precautions
1. AM esters are hygroscopic. Ensure the product reaches room temperature before opening after removal from the refrigerator. Fluo-8 AM is present in trace amounts and may be difficult to observe; briefly centrifuge after warming to ensure the powder collects at the bottom of the tube.
2. Fluo-8 AM decomposes rapidly upon contact with water. If not used in one batch, aliquot the stock solution for storage.
3. All fluorescent dyes are subject to photobleaching; avoid light exposure as much as possible.
4. This product is for research use only and shall not be stored in ordinary residential premises.
5. For your safety and health, comply with the general laboratory safety regulations of your institution.
Instructions for Use
I. Pre-Experiment Preparation
1. Stock Solution Preparation
Remove Fluo-8 AM from the refrigerator, allow it to reach room temperature, and briefly centrifuge to collect the powder at the tube bottom. Prepare a 2-5 mM Fluo-8 AM stock solution using anhydrous DMSO. Example: To prepare a 5 mM stock solution, add 191 µL of anhydrous DMSO to a 1 mg vial.
Note: Aliquot the prepared stock solution and store at -20 °C; tightly seal the tube caps with parafilm for optimal preservation.
2. Working Solution Preparation
(1) Dilute the Fluo-8 AM stock solution with PBS or HBSS to prepare a 4 μM working solution. A final concentration of 4-5 μM is recommended for most cell lines.Note: Optimal loading concentrations vary by cell type. Perform a concentration optimization assay before formal experiments. To avoid cytotoxicity from overloading, use the lowest effective probe concentration, starting with 4 μM.
(2) (Optional) If Fluo-8 AM uptake is inefficient, add an appropriate amount of 20% Pluronic F-127 solution to the Fluo-8 AM working solution to prevent aggregation in the buffer and enhance cellular uptake. Maintain the final concentration of Pluronic F-127 at 0.04-0.05%.
Notes:(1) Preparation of 20% (w/v) Pluronic F-127 DMSO Stock Solution: Dissolve 100 mg of Pluronic F-127 in 0.5 mL of DMSO. Heat at 40-50 °C for 20-30 minutes to dissolve completely. Store at room temperature (do not refrigerate). If crystallization occurs, reheat to redissolve; this does not affect performance.
(2) Pluronic F-127 may reduce the stability of Fluo-8 AM. Add it only to the working solution, not the stock solution.
(3) If your cells express organic anion transporters, add probenecid (1-2 mM stock) to the dye working solution (final concentration 0.5-1 mM) to reduce leakage of the de-esterified indicator.
3. Instrument Preparation
(1) Fluorescence microscope (Ex/Em: 494/516 nm)
(2) Flow cytometer (Ex/Em: 494/516 nm)
(3) Fluorescent microplate reader (Ex/Em: 494/516 nm)
4. Control Group Setup
| Group | Fluo-8 AM Calcium Probe | Sample Cells | | Negative Control | + | Untreated cells | | Experimental Group | + | Experimentally treated cells |
|
(1) Negative Control Group: Cells without any treatment, serving as a reference for calcium signal changes in the experimental group after treatment.
(2) Experimental Group: To verify probe activity and cell loading efficiency.
II. Operating Procedures (96-Well Plate Format)
Protocol 1: Fluorescence Microscopy (for Suspension Cells)
1. Cell Harvesting and Washing
(1) Collect target suspension cells into centrifuge tubes, centrifuge at 1000 rpm for 5 minutes at room temperature, and carefully aspirate the supernatant.
(2) Wash the cells once with PBS or HBSS (or other standard buffers).
Note: Washing typically reduces background fluorescence. Use a vacuum pump for aspirating culture medium, PBS, or HBSS. Phenol red or serum may interfere with the assay.
2. Cell Resuspension and Counting
(1) Resuspend the cell pellet in PBS or HBSS and perform cell counting.
(2) Take 5×10⁴-10⁵ cells, centrifuge at 1000 rpm for 5 minutes at low speed, and remove the PBS/HBSS.
Note: Cell density is not critical for microscopy, but must be consistent between the experimental and control groups. Adjust flexibly based on sample type and experimental conditions; optimal confluency is 70-85% in the microscopic field.
3. Cell Staining
(1) Add 100 µL of Fluo-8 staining working solution to each well of the 96-well plate. Incubate at 37 °C for 30 minutes in the dark. Incubation time can be adjusted between 10-60 minutes; rapid staining is achievable in 10 minutes to maximize cell viability and simplify the workflow.Note: For first-time use, incubate at 37 °C for 30 minutes to evaluate fluorescence performance. If excessive cell death occurs, shorten the incubation time; if fluorescence intensity is too low, extend the incubation time.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Microscopic Observation and ImagingTransfer the cell suspension to the 96-well plate, allow the cells to settle and adhere to the bottom, and observe under a fluorescence microscope.
Note: Adjust the volume of the cell suspension based on cell count. Optimal cell distribution in the microscopic field is 70-85%.
Protocol 2: Fluorescence Microscopy (for Adherent Cells)
1. Cell Seeding
Seed cells in a 96-well plate one day in advance to achieve 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 PBS or HBSS to gently wash the cells once; completely aspirate the PBS/HBSS after washing.
3. Cell Staining
(1) Add 100 µL of Fluo-8 staining working solution to each well. Incubate at 37 °C for 30 minutes in the dark. Incubation time can be adjusted between 10-60 minutes; rapid staining is achievable in 10 minutes to maximize cell viability and simplify the workflow.
Note: For first-time use, incubate at 37 °C for 30 minutes to evaluate fluorescence performance. If excessive cell death occurs, shorten the incubation time; if fluorescence intensity is too low, extend the incubation time.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Microscopic Observation and Imaging
(1) After washing, add an appropriate volume of PBS or HBSS to each well to keep the cells moist.
(2) Place the plate directly under a fluorescence microscope for observation and imaging. Use the same filter settings as in Protocol 1.
Protocol 3: Flow Cytometry (for Suspension and Adherent Cells)
1. Cell Preparation
(1) Suspension Cells: Follow the steps in Protocol 1.
(2) Adherent Cells:
a. Aspirate the culture medium and gently wash the cells once with PBS or HBSS.
b. Add an appropriate amount of trypsin digestion solution (covering the cells) and incubate at room temperature. Observe under a microscope; once cells round up and intercellular spaces increase, gently pipette to detach cells completely.
c. Critical Step: Immediately add serum-containing complete medium to terminate digestion.
d. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, and aspirate the supernatant.
e. Resuspend the cells in PBS or HBSS and perform cell counting.
f. Take 10⁶ cells, centrifuge at 1000 rpm for 5 minutes at low speed at room temperature, and aspirate the supernatant.
2. Cell Staining
(1) Add 1 mL of Fluo-8 staining working solution to the cells. Incubate at 37 °C for 30 minutes in the dark. Incubation time can be adjusted between 10-60 minutes; rapid staining is achievable in 10 minutes to maximize cell viability and simplify the workflow.Note: For first-time use, incubate at 37 °C for 30 minutes to evaluate fluorescence performance. If excessive cell death occurs, shorten the incubation time; if fluorescence intensity is too low, extend the incubation time.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
3. Flow Cytometry Analysis
(1) Important: Perform flow cytometry analysis within 1 hour of staining to ensure stable fluorescence signals.
(2) Analyze samples on a flow cytometer using the pre-set detection channel.
(3) First, adjust the voltage using blank controls (unstained cells) to position the cell population in the lower-left corner of the coordinate system.
(4) Next, detect the control and experimental groups to confirm the validity of the experimental system.
(5) Finally, detect the experimental samples and record/analyze the mean fluorescence intensity (MFI) of the cells.
Note: All stained samples must be kept on ice in the dark and analyzed immediately. Quantitative experiments (e.g., flow cytometry) must be completed strictly within 1 hour to ensure accurate and reproducible data.
Protocol 4: Fluorescent Microplate Reader (for Suspension and Adherent Cells)
1. Cell Seeding
Seed cells in a black 96-well microplate. The number of cells per well should be 100–10,000; a range of 2,000-5,000 cells is typically recommended.
2. Cell Washing
Collect the target cell suspension into centrifuge tubes.
(1) Suspension Cells: Centrifuge at 250-1000 × g for 5 minutes at room temperature, aspirate the supernatant, and wash once with PBS or HBSS (or other standard buffers).
(2) Adherent Cells: Aspirate the culture medium and wash the cells once with PBS or HBSS (or other standard buffers).
Note: Washing typically reduces background fluorescence. Use a vacuum pump for aspirating culture medium and PBS/HBSS. Phenol red or serum may interfere with the assay.
3. Cell Staining
(1) Add 100 µL of Fluo-8 staining working solution to each well. Incubate at 37 °C for 30 minutes in the dark. Incubation time can be adjusted between 10-60 minutes; rapid staining is achievable in 10 minutes to maximize cell viability and simplify the workflow.Note: For first-time use, incubate at 37 °C for 30 minutes to evaluate fluorescence performance. If excessive cell death occurs, shorten the incubation time; if fluorescence intensity is too low, extend the incubation time.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Fluorescent Microplate Reader Detection
Detect samples using the pre-set parameters on the fluorescent microplate reader. The effect of drug stimulation can be determined by comparing the Relative Fluorescence Units (RFU) of the control and treatment groups.
III. Result Interpretation
1. Qualitative Analysis (Microscopy)
Figure 1. Dynamic Changes in Intracellular Calcium Ion Concentration in SH-SY5Y Cells
Under a fluorescence microscope:
Figure 1 compares the fluorescence intensities of Fluo (calcium signal) and DCFDA (ROS signal) in different treatment groups, enabling systematic analysis of the synergistic or independent effects of various stimuli on cellular calcium signaling pathways and oxidative stress.
Note: Image adapted from Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release (doi:10.1038/s41598-023-38210-w).
Frequently Asked Questions (FAQs)
1. Q: Why is no fluorescence detected, or is the fluorescence intensity too low?
A: This may be due to insufficient cell density; increase the cell density and consider extending the incubation time.
2. Q: What should I do if excessive cell death is observed after Fluo-8 incubation?
A: First, evaluate whether the dye concentration and treatment time are appropriate. Although Fluo-8 AM is relatively safe, excessively high concentrations or prolonged staining may cause cell damage. Reduce the dye concentration or shorten the staining time, and closely monitor cell status during the experiment.
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