Fluo-20 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-20 AM钙离子荧光探针为核心检测成分，其检测原理如下：Fluo-20 AM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-20，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-20 AM作为经典钙指示剂Fluo-8 AM、Fluo-4 AM、Fluo-3 AM的升级产品，在性能上实现全面优化。Fluo-3AM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-20 AM可在室温条件下进行细胞染色，Fluo-3AM和Fluo-4 AM则必须在 37 ℃的环境中才能进行细胞上样操作。Fluo-20 AM在显著改善了细胞负载情况以及钙反应效果。Fluo-20 AM在荧光亮度上是Fluo-8 AM的 2 倍，Fluo-4 AM的 4 倍，是Fluo-3 AM的 8 倍，荧光亮度远超Fluo-8 AM、Fluo-4AM和Fluo-3 AM。
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4倍、Fluo-3 AM的8倍、Fluo-8 AM的2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、Ex/Em：494/516 nm；
3、光谱图：
产品组分：
| 组分 | 2300 T | | Fluo-20 AM Calcium Probe | 5 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-20 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-20 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-20 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-20 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-20 AM储液制备4 μM的Fluo-20 AM工作液。对于大多数细胞系，建议使用终浓度为4-5μM的Fluo-20 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-20 AM进入细胞的效果不好，可向Fluo-20 AM溶液中加入适量20% Pluronic F-127溶液，防止Fluo-20AM在缓冲液中聚集并促进Fluo-20 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-20 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-20 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-20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-20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，取5×10⁴-10⁵个细胞。
(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-20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) 流式细胞用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洗涤1次。
(2) 贴壁细胞：吸除培养液，用PBS或HBSS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，用PBS或HBSS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-20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：
定性分析 (显微镜)：
图1. 神经元细胞钙离子浓度动态变化图
在荧光显微镜下：
图1为在荧光显微镜下，观察不同时间点细胞内绿色荧光的分布以及强度情况，分析神经元在刺激下的钙信号动态响应过程，研究钙信号在神经元功能中的作用机制。
注：图片引自《Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis,and maturation》(doi:10.1126/sciadv.aaz9499.)
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-20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-20 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-20 AM Calcium Probe is a professional tool for the precise detection of intracellular calcium ion concentration changes. This product uses the Fluo-20 AM calcium ion fluorescent probe as its core detection component, and its detection principle is as follows: Fluo-20 AM is cell membrane-permeable and can easily enter cells with simple incubation. Once inside the cell, it is cleaved by intracellular esterases to generate membrane-impermeable Fluo-20, which is retained in the cell to exert its corresponding physiological functions. This product enables the detection of calcium ion concentration changes on multiple platforms, including fluorescence microscopes, fluorescence microplate readers, and flow cytometers, providing key technical support for cell biology research, disease mechanism exploration, and drug development.
As an upgraded version of the classic calcium indicators Fluo-8 AM, Fluo-4 AM, and Fluo-3 AM, Fluo-20 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 research; however, they only exhibit moderate fluorescence intensity in live cells, and relatively harsh cell loading conditions are required to achieve optimal cellular calcium responses. In contrast, Fluo-20 AM allows cell staining at room temperature, whereas Fluo-3 AM and Fluo-4 AM must be loaded onto cells at 37 °C. Fluo-20 AM significantly improves cell loading and calcium response outcomes. Its fluorescence brightness is twice that of Fluo-8 AM, four times that of Fluo-4 AM, and eight times that of Fluo-3 AM, far exceeding the fluorescence brightness of Fluo-8 AM, Fluo-4 AM, and Fluo-3 AM.
Application: Calcium ion detection
Product Features:
1. 10-minute rapid staining: Directly add the reagent after mixing, and staining can be completed in as little as 10 minutes after incubation, greatly reducing experimental time and operational steps.
2. Flexible loading conditions: Excellent staining can be achieved at either room temperature or 37 °C, eliminating the need for harsh cell loading conditions.
3. Higher fluorescence intensity: Fluorescence brightness is 4 times that of Fluo-4 AM, 8 times that of Fluo-3 AM, and 2 times that of Fluo-8 AM, significantly improving the distinguishability of detection signals.
4. Ultra-high sensitivity: Low background signal interference, high detection sensitivity, and easy capture of low-amplitude calcium signal fluctuations.
Product Parameters:
1. Appearance: Solid powder soluble in DMSO.
2. Ex/Em: 494/516 nm.
3. Spectrum:
Product Components:
Component
| 2300 T | | Fluo-20 AM Calcium Probe | 5 mg |
|
Note: Calculated based on 100 μL of staining working solution per use, with a working solution concentration of 4 μM.
Precautions:
1. AM ester groups are hygroscopic. Ensure the product is equilibrated to room temperature before opening. Fluo-20 AM is a trace amount and difficult to observe; after equilibration to room temperature, perform a quick spin to ensure the powder is centrifuged to the bottom of the tube.
2. Fluo-20 AM is extremely sensitive to hydrolysis in aqueous solutions. If not used entirely at once, it is recommended to aliquot the stock solution into small portions for storage.
3. All fluorescent dyes are subject to photobleaching; minimize exposure to light.
4. This product is for research use only and must not be stored in ordinary residential premises.
5. For your safety and health, please comply with the standard laboratory safety regulations of your institution.
Instructions for Use:
I. Pre-Experiment Preparation
1. Stock Solution Preparation:
Remove Fluo-20 AM from the refrigerator, equilibrate to room temperature, and perform a quick spin to pellet the powder at the bottom of the tube. Prepare a 2-5 mM Fluo-20 AM stock solution using anhydrous DMSO. For example: To prepare a 5 mM stock solution, add 191 µL of anhydrous DMSO to a tube containing 1 mg of Fluo-20 AM.
Note: The prepared stock solution should be aliquoted and stored at -20 °C, preferably with Parafilm tightly wrapped around the tube cap.
2. Working Solution Preparation:
(1) Dilute the Fluo-20 AM stock solution with PBS or HBSS to prepare a 4 μM Fluo-20 AM working solution. For most cell lines, a final working concentration of 4-5 μM is recommended.
Note: The optimal loading concentration varies by cell type. It is recommended to determine the optimal concentration before formal experiments. To avoid cytotoxicity from overloading, use the lowest probe concentration that yields valid results, starting with 4 μM for optimization.
(2) (Optional) If Fluo-20 AM uptake into cells is poor, add an appropriate amount of 20% Pluronic F-127 solution to the Fluo-20 AM solution to prevent aggregation in the buffer and promote cellular uptake. The final concentration of Pluronic F-127 should be controlled between 0.04–0.05%.
Note: (1) To prepare a 20% (w/v) Pluronic F-127 DMSO stock solution: Add 0.5 mL of DMSO to 100 mg of Pluronic F-127. Dissolution requires heating at 40-50 °C for 20-30 minutes. Store at room temperature after dissolution; do not refrigerate. If crystals precipitate, reheat to dissolve before use, which does not affect performance.
(2) The use of Pluronic F-127 reduces the stability of Fluo-20 AM. Therefore, it is only recommended to add it when preparing the working solution, not the stock solution.
(3) If your cells express organic anion transporters, add probenecid (1-2 mM) 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) Fluorescence microplate reader, Ex/Em: 494/516 nm.
4. Control Setup:
| Group | Fluo-20 AM Calcium Probe | Sample Cells | | Negative Control | + | Untreated cells | | Experimental Group | + | Experimentally treated cells |
|
(1) Negative control: Cells are not subjected to any treatment, providing a reference for calcium signal changes after experimental treatment in the test group.
(2) Experimental group: To verify probe activity and cell loading efficiency.
II. Operating Procedures (Using a 96-well Plate as an Example)
Protocol 1: Fluorescence Microscopy Detection (Suspension Cells)
1. Cell Collection and Washing:
(1) Collect the test suspension cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, and carefully aspirate the supernatant.
(2) Wash the cells once with a conventional buffer such as PBS or HBSS.
Note: Washing generally reduces background fluorescence. When aspirating culture medium, PBS, HBSS, or other conventional buffers, a vacuum pump is preferred. Phenol red or serum may interfere with the detection of this kit.
2. Cell Resuspension and Counting:
(1) Resuspend the cell pellet in PBS or HBSS and perform cell counting.
(2) Take 5×10⁴-1×10⁵ cells, centrifuge at 1000 rpm for 5 minutes at low speed, and remove PBS or HBSS.
Note: The microscopy method is not strict on cell density, as long as the density is consistent between the experimental and control groups. It can be flexibly adjusted according to sample type and experimental conditions, with 70–85% cell distribution in the microscope field of view being optimal.
3. Cell Staining:
(1) Add 100 µL of Fluo-20 staining working solution to the 96-well plate and incubate at 37 °C in the dark for 30 minutes. The incubation time can be adjusted between 10-60 minutes, with rapid staining achievable in as little as 10 minutes to maximize cell viability and simplify the experimental workflow.
Note: If the incubation temperature and time are uncertain for the first experiment, it is recommended to incubate at 37 °C for 30 minutes initially and observe the fluorescence effect. If excessive cell death occurs, shorten the time appropriately; if the fluorescence intensity is too weak, extend the time appropriately.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Microscopic Observation and Photography:
Add the cell suspension to the 96-well plate, let it stand briefly to allow the cells to settle naturally to the bottom, and observe under a fluorescence microscope.
Note: The volume of cell suspension can be adjusted according to the cell count, with 70-85% cell distribution in the microscope field of view being optimal.
Protocol 2: Fluorescence Microscopy Detection (Adherent Cells)
1. Cell Treatment:
Seed cells in a 96-well plate one day in advance to achieve 70-85% confluency. After the 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, then aspirate the PBS or HBSS completely after washing.
3. Cell Staining:
(1) Add 100 µL of Fluo-20 staining working solution to the 96-well plate and incubate at 37 °C in the dark for 30 minutes. The incubation time can be adjusted between 10-60 minutes, with rapid staining achievable in as little as 10 minutes to maximize cell viability and simplify the experimental workflow.
Note: If the incubation temperature and time are uncertain for the first experiment, it is recommended to incubate at 37 °C for 30 minutes initially and observe the fluorescence effect. If excessive cell death occurs, shorten the time appropriately; if the fluorescence intensity is too weak, extend the time appropriately.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Microscopic Observation and Photography:
(1) After washing, add an appropriate volume of PBS or HBSS to each well to keep the cells moist.
(2) Place the culture plate directly under the fluorescence microscope for observation and photography. Filter settings are the same as in Protocol 1.
Protocol 3: Flow Cytometry Detection (Suspension and Adherent Cells)
1. Cell Preparation:
(1) Suspension cells: Same as Protocol 1; take 5×10⁴-1×10⁵ cells.
(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 (just enough to cover the cells), observe under a microscope at room temperature until the cells round up and intercellular spaces increase, then gently pipette to detach the cells completely.
c. Critical step: Immediately add complete medium containing serum 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 1×10⁶ 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-20 staining working solution to the cells and incubate at 37 °C in the dark for 30 minutes. The incubation time can be adjusted between 10-60 minutes, with rapid staining achievable in as little as 10 minutes to maximize cell viability and simplify the experimental workflow.
Note: If the incubation temperature and time are uncertain for the first experiment, it is recommended to incubate at 37 °C for 30 minutes initially and observe the fluorescence effect. If excessive cell death occurs, shorten the time appropriately; if the fluorescence intensity is too weak, extend the time appropriately.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
3. Instrument Detection:
(1) Important: Perform flow cytometry detection within 1 hour after staining to ensure stable fluorescence signals.
(2) Analyze using a flow cytometer under preset detection channels.
(3) First, adjust the voltage using a blank control (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 and analyze the mean fluorescence intensity of the cells.
Note: All samples must be kept in the dark, on ice, and detected immediately after staining. Quantitative experiments (e.g., flow cytometry) must be completed strictly within 1 hour to ensure accurate and reproducible data.
Protocol 4: Fluorescence Microplate Reader Detection (Suspension and Adherent Cells)
1. Seeding and Culture:
Seed cells in a black 96-well plate, controlling the number of cells per well between 100-10,000, typically 2000-5000.
2. Washing:
Collect the test suspension cell suspension into a centrifuge tube.
(1) Suspension cells: Centrifuge at 250-1000× g for 5 minutes at room temperature, aspirate the supernatant, and wash once with PBS or HBSS.
(2) Adherent cells: Aspirate the culture medium and wash the cells once with PBS or HBSS.
Note: Washing generally reduces background fluorescence. When aspirating culture medium, PBS, or HBSS, a vacuum pump is preferred. Phenol red or serum may interfere with the detection of this kit.
3. Cell Staining:
(1) Add 100 µL of Fluo-20 staining working solution to the 96-well plate and incubate at 37 °C in the dark for 30 minutes. The incubation time can be adjusted between 10-60 minutes, with rapid staining achievable in as little as 10 minutes to maximize cell viability and simplify the experimental workflow.
Note: If the incubation temperature and time are uncertain for the first experiment, it is recommended to incubate at 37 °C for 30 minutes initially and observe the fluorescence effect. If excessive cell death occurs, shorten the time appropriately; if the fluorescence intensity is too weak, extend the time appropriately.
(2) After incubation, wash the cells 1-3 times with PBS or HBSS.
4. Fluorescence Microplate Reader Detection:
Detect under the preset conditions of the fluorescence microplate reader. By comparing the RFU (Relative Fluorescence Units) of the control and treatment groups, the effect of drug stimulation can be determined.
III. Result Interpretation:
Qualitative Analysis (Microscopy):
Figure 1. Dynamic changes in intracellular calcium concentration in neuronal cells
Under a fluorescence microscope:
Figure 1 shows the distribution and intensity of green fluorescence within cells at different time points, analyzed to study the dynamic calcium response of neurons under stimulation and the mechanism of calcium signaling in neuronal function.
Note: Image adapted from "Bioengineered perfused human brain microvascular networks enhance neural progenitor cell survival, neurogenesis, and maturation" (doi:10.1126/sciadv.aaz9499).
Frequently Asked Questions and Answers:
1. Q: Why is no fluorescence value detected or the fluorescence value is low?
A: This may be due to insufficient cell density; you can increase the cell density. Alternatively, consider extending the incubation time.
2. Q: What should I do if excessive cell death is observed after Fluo-20 incubation?
A: First, assess whether the dye concentration and treatment time are appropriate. Although Fluo-20 AM is relatively safe, excessively high concentrations or prolonged staining times may still cause cell damage. It is recommended to reduce the dye concentration or shorten the staining time, and closely monitor cell status during the experiment. Note 2:
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