计算溶液所需的质量、体积或浓度。
Fluo-3钙离子检测试剂盒
有货
为什么选择此级别
BioReagent,生物染色剂,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。
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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
Fluo-3钙离子检测试剂盒 (Fluo-3 Calcium Ion Detection Kit)是一种基于Fluo-3 AM钙离子荧光探针检测细胞内钙离子浓度的试剂盒。本试剂盒可以使用荧光显微镜进行荧光成像检测，也可以使用荧光酶标仪或流式细胞仪进行荧光的定量检测。使用荧光显微镜或荧光酶标仪还可以进行细胞内钙离子浓度的动态变化检测。
钙 (Calcium)通常是哺乳动物机体内最为丰富的矿物质，参与调控众多的细胞生命活动过程，也是最为重要的细胞内调控因子之一。钙能够以自由离子或结合钙离子的复合物两种形式存在，例如磷酸钙和碳酸钙复合物即为骨骼组织的组成成分。包括肌肉收缩、细胞粘附、激素/神经递质释放、糖原代谢、细胞增殖/分化、血凝、神经或突触传递以及骨骼结构支持在内的大量生理过程都受到钙离子信号的调控。细胞特异性钙信号系统完整性的缺陷会导致一些疾病的发生[1]。
本试剂盒检测细胞内钙离子流通量的原理是：钙离子荧光探针Fluo-3 AM本身几乎无荧光，一旦进入细胞内经过胞内酯酶水解后产生的Fluo-3和钙离子结合后可以发出绿色荧光。细胞外的钙离子内流，或者细胞内储存的钙离子释放，使得细胞浆内的钙离子浓度增加，细胞内的Fluo-3可以结合更多的钙离子，最终引起细胞内绿色荧光强度增加；细胞内的钙离子外流或储存，使得细胞浆内的钙离子浓度降低，细胞浆内的Fluo-3 可以结合的钙离子减少，最终引起细胞内绿色荧光强度下降。钙离子荧光探针Fluo-3 AM可广泛地用于钙离子浓度的动态变化检测，以及高通量筛选影响钙离子浓度变化的G蛋白偶联受体激动剂或抑制剂等。
本试剂盒使用便捷，提供了多种配套试剂，并且染色后无需洗涤。本试剂盒提供了用于稀释Fluo-3 AM的检测缓冲液 (Assay Buffer)，该检测缓冲液可以极大程度地保证细胞活力和接近正常的生长状态；提供了可增强Fluo-3 AM溶解性的促溶剂 (Solubility Enhancer)可以改善染色效果；提供的染色增强剂 (Staining Enhancer)在检测中可有效地维持细胞内荧光并减少背景荧光。同时，本试剂盒染色后可不用洗涤，使用更方便。
对于96孔板中的样品，按照每孔使用100 μL Fluo-3染色液计算，本试剂盒的包装可以进行200次检测；如果用于流式细胞仪检测，按照每个样品的检测体系体积为0.5 ml时，本试剂盒的包装可以进行40次检测；对于6孔板中的贴壁培养细胞样品，按照每孔使用1 ml Fluo-3染色液计算，本试剂盒的包装可以进行20次检测。
注意事项:
1. Fluo-3 AM遇水极易分解，建议第一次使用时，适当酌情分装。
2. Fluo-3 AM在4℃、冰浴等较低温度情况下会凝固而粘在离心管管底、管壁或管盖内，可以20-25℃水浴温育片刻至全部融解并适当离心至管底后使用。
3. Fluo-3 AM的工作浓度、细胞量、孵育温度和时间等可根据所使用的细胞和给予的特定刺激预先进行适当摸索和优化，例如荧光探针的浓度可在 0.2×-2×之间适当调整。
4. Fluo-3 AM (500×)和Solubility Enhancer (500×)使用时请确保已完全溶解。
5. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
6. 本公司所有产品仅限于专业人员用于生命科学研究，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅。
7. 本公司所有产品必须由合格专业技术人员操作同时佩戴口罩/手套/实验服并遵守生物实验室安全操作规程。
使用方法：
1. Fluo-3染色液的配制：
按照96孔板每孔100 μL Fluo-3染色液 (Fluo-3 Staining Solution)的体系，参考下表配制适量的Fluo-3染色液，并充分混匀。
| 试剂 | 1 Sample | 10 Samples | 100 Samples |
| Fluo-3 AM | 0.2 μL | 2 μL | 20 μL |
| 增溶剂 (500×) | 0.2 μL | 2 μL | 20 μL |
| 检测缓冲液 | 99.6 μL | 996 μL | 9.96 mL |
| Fluo-3染色液 | 100 μL | 1 mL | 10 mL |
注1：配制好的Fluo-3染色液必须一次使用完，不能冻存。Fluo-3染色液中Fluo-3 AM的浓度可以根据染色效果在0.2×-2×之间适当调整。
注2：也可以使用其它合适的溶液，如无血清培养液、HBSS或PBS稀释Fluo-3 AM (500×)。本试剂盒配套提供的检测缓冲液 (Assay Buffer)可在一段时间内有效维持细胞的正常状态，并给细胞提供一定的营养，使用效果通常比PBS或HBSS更好。
注3：Staining Enhancer即染色增强剂，对细胞功能可能有一定的影响，对于大多数细胞无需添加。如果遇到细胞 (例如CHO)出现去酯化的荧光探针Fluo-3明显的外排现象时，则建议添加。使用时将Staining Enhancer (100×)按照1: 100稀释到Fluo-3 Staining Solution中，配制成含1× Staining Enhancer的Fluo-3 Staining Solution。细胞在含1× Staining Enhancer的溶液中孵育的时间不能超过2小时。
2. 荧光显微镜检测：
(1) 接种培养。将细胞接种于96孔板等多孔板、细胞培养皿中或者细胞爬片上。如有必要，按实验设计对细胞进行一定处理。
(2) 洗涤 (选做)。对于贴壁细胞，吸除培养液，用PBS洗涤细胞1遍；对于悬浮细胞，250-1000 ×g 室温离心5 min，吸除上清，用PBS洗涤1遍。酚红或血清对于本试剂盒的检测有一定的干扰，吸除培养液和PBS时最好使用真空泵。在能充分吸净残留液体的情况下，可以不使用PBS洗涤。 通常洗涤一次能更好地降低背景荧光，但本试剂盒不进行洗涤也能获得良好染色效果。
(3) 染色。加入适当体积的Fluo-3染色液。通常96孔板每孔加入100 μL，24孔板每孔加入250 μL，12孔板每孔加入500 μL，6孔板每孔加入1 ml。37℃避光孵育30 min。孵育时间可在10-60 min之间进行调整。
注1：如果是首次实验不能确定孵育温度和时间，建议先尝试37℃孵育30 min，观察荧光效果。如果细胞死亡较多，则适当缩短时间或降低温度；如果荧光强度太弱，则适当延长时间。
注2：孵育后也可用PBS或HBSS洗涤1-3次。
(4) 检测。孵育结束后，在荧光显微镜下观察染色效果 (Fluo-3 AM为绿色荧光，Ex/Em=490/525nm)。
3. 流式细胞仪检测：
(1) 细胞准备。贴壁细胞胰酶消化后用培养液重悬，并用PBS洗涤一次；悬浮细胞250-1000 ×g室温离心 5 min，弃上清，用PBS洗涤一次。每个样品推荐的细胞用量为10⁶个细胞。
(2) 染色。对于上一步骤的10⁶个细胞的沉淀，加入1 ml Fluo-3染色液，重悬为单细胞悬液。37℃避光孵育 30 min。 孵育时间可在10-60 min 之间进行调整。
注1：如果是首次实验不能确定孵育温度和时间，建议先尝试 37℃孵育30 min，观察荧光效果。如果细胞死亡较多，则适当缩短时间或降低温度；如果荧光强度太弱，则适当延长时间。
注2：孵育后也可用PBS或HBSS洗涤1-3次。
(3) 检测。孵育完成后，可以直接进行流式细胞仪检测，也可以250-1000× g室温离心5 min沉淀细胞，吸净液体后每个样品加入0.5 ml检测缓冲液重悬细胞后用流式细胞仪检测 (Fluo-3 AM为绿色荧光，Ex/Em=490/525nm)。
注1：使用仅含检测缓冲液的并且未经染色的细胞样品用于流式细胞仪的阴性对照设置。
注2：由于流式检测比较灵敏，使用的荧光探针浓度可能要比荧光显微镜检测时要低，此时可根据细胞类型和实际染色情况对Fluo-3 AM稀释倍数进行适当调整。
4.荧光酶标仪检测：
(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100-10,000个，通常宜在 2000-5000 范围内。按实验设计对细胞进行一定处理。
(2) 洗涤 (选做)。对于贴壁细胞，吸除培养液，用PBS洗涤细胞1遍；对于悬浮细胞，250-1000× g室温离心 5 min，吸除上清，用PBS洗涤1遍。酚红或血清对于本试剂盒的检测有一定的干扰，吸除培养液和PBS时最好使用真空泵。在能充分吸净残留液体的情况下，可以不使用PBS洗涤。通常洗涤一次能更好地降低背景荧光，但本试剂盒不进行洗涤也能获得良好染色效果。
(3) 染色。加入适当体积的Fluo-3染色液，通常96孔板每孔加入100 μL。37℃避光孵育30 min。孵育时间可在10-60 min之间进行调整。
注1：如果是首次实验不能确定孵育温度和时间，建议先尝试37℃孵育30 min，观察荧光效果。如果细胞死亡较多，则适当缩短时间或降低温度；如果荧光强度太弱，则适当延长时间。
注2：孵育后也可用PBS或HBSS洗涤1-3次。
(4) 检测。孵育结束后，用荧光酶标仪检测 (Fluo-3 AM为绿色荧光，Ex/Em=490/525nm)。
通过对比对照组与处理组的RFU (Relative fluorescence values)，可以得出药物刺激的效果。
参考文献：
1.Clapham DE. Cell. 2007. 131(6):1047-58.
2.Wu N, Nishioka WK, Derecki NC, Maher MP. Sci Rep. 2019. 9(1):12692.
3.Hagen BM, Boyman L, Kao JP, Lederer WJ. Cell Calcium. 2012. 52(2):170-81.
Fluo‑3 Calcium Ion Detection Kit is a kit for detecting intracellular calcium ion concentration based on the Fluo‑3 AM calcium fluorescent probe. This kit can be used for fluorescence imaging via fluorescence microscopy, as well as for quantitative fluorescence detection using a fluorescence microplate reader or flow cytometry. Dynamic changes in intracellular calcium ion concentration can also be detected using fluorescence microscopy or a fluorescence microplate reader.
Calcium is generally the most abundant mineral in mammalian organisms, involved in the regulation of numerous cellular processes and is one of the most important intracellular regulatory factors. Calcium exists either as free ions or in calcium‑bound complexes; for instance, calcium phosphate and calcium carbonate complexes are major components of bone tissue. A wide range of physiological processes are regulated by calcium signaling, including muscle contraction, cell adhesion, hormone/neurotransmitter release, glycogen metabolism, cell proliferation/differentiation, blood coagulation, neuronal or synaptic transmission, and structural support of bone. Defects in the integrity of cell‑specific calcium signaling systems can lead to the development of various diseases[1].
The principle of this kit for detecting intracellular calcium flux is as follows: the calcium fluorescent probe Fluo‑3 AM itself exhibits almost no fluorescence. Once inside cells, it is hydrolyzed by intracellular esterases to form Fluo‑3, which emits green fluorescence upon binding calcium ions. Influx of extracellular calcium or release of calcium from intracellular stores increases cytosolic calcium concentration, allowing more calcium binding to intracellular Fluo‑3 and ultimately enhancing intracellular green fluorescence intensity. Efflux or sequestration of intracellular calcium reduces cytosolic calcium concentration, decreasing calcium available for Fluo‑3 binding and thereby lowering green fluorescence intensity. The calcium fluorescent probe Fluo‑3 AM is widely used for detecting dynamic changes in calcium concentration and for high‑throughput screening of G‑protein‑coupled receptor agonists or antagonists that affect calcium mobilization.
This kit is convenient to use and provides a complete set of supporting reagents, with no washing required after staining. The kit includes Assay Buffer for diluting Fluo‑3 AM, which greatly preserves cell viability and near‑normal growth conditions. It also provides Solubility Enhancer to improve the solubility of Fluo‑3 AM and enhance staining performance. The included Staining Enhancer effectively retains intracellular fluorescence and reduces background fluorescence during detection. Meanwhile, no washing step is needed after staining, making the kit very convenient to use.
For samples in 96‑well plates, the kit package supports 200 tests based on 100 μL Fluo‑3 Staining Solution per well. For flow cytometry assays with a 0.5 mL detection system per sample, the kit supports 40 tests. For adherent cultured cells in 6‑well plates, the kit supports 20 tests based on 1 mL Fluo‑3 Staining Solution per well.
Precautions
1. Fluo‑3 AM is highly unstable in water; appropriate aliquots are recommended upon first use.
2. Fluo‑3 AM may solidify at low temperatures such as 4 ℃ or on ice and adhere to the bottom, wall, or cap of the centrifuge tube. Warm briefly in a 20–25 ℃ water bath until fully dissolved, centrifuge briefly to collect at the bottom, then use.
3. The working concentration of Fluo‑3 AM, cell amount, incubation temperature, and time can be optimized according to the cell type and specific stimuli applied. For example, the probe concentration can be adjusted appropriately between 0.2× and 2×.
4. Ensure Fluo‑3 AM (500×) and Solubility Enhancer (500×) are completely dissolved before use.
5. All fluorescent dyes are subject to photobleaching; protect from light as much as possible to slow fluorescence decay.
6. All products of this company are for research use only by professional personnel in life science research. They are not intended for clinical diagnosis or therapy, food or drug applications, or storage in ordinary residential premises.
7. All products must be operated by qualified professional technicians wearing masks, gloves, and lab coats, in compliance with biological laboratory safety protocols.
Protocol
1. Preparation of Fluo‑3 Staining Solution
Prepare an appropriate volume of Fluo‑3 Staining Solution according to the system of 100 μL per well in 96‑well plates, referring to the table below, and mix thoroughly.
| Reagent | 1 Sample | 10 Samples | 100 Samples |
| BioFluo-3 AM | 0.2 μL | 2 μL | 20 μL |
| Solubility Enhancer (500×) | 0.2 μL | 2 μL | 20 μL |
| Assay Buffer | 99.6 μL | 996 μL | 9.96 mL |
| BioFluo-3 Staining Solution | 100 μL | 1 mL | 10 mL |
Note 1: Prepared Fluo‑3 Staining Solution must be used immediately in one run and cannot be frozen. The concentration of Fluo‑3 AM in the staining solution can be adjusted between 0.2× and 2× based on staining performance.
Note 2: Other suitable solutions such as serum‑free medium, HBSS, or PBS may also be used to dilute Fluo‑3 AM (500×). The Assay Buffer provided with this kit effectively maintains normal cell status and provides partial nutrition over time, generally yielding better results than PBS or HBSS.Note 3: Staining Enhancer may have certain effects on cell function and is unnecessary for most cells. It is recommended for cells (e.g., CHO) showing obvious extrusion of de‑esterified fluorescent Fluo‑3. Add Staining Enhancer (100×) to Fluo‑3 Staining Solution at a dilution of 1:100 to prepare solution containing 1× Staining Enhancer. Cell incubation time in solution containing 1× Staining Enhancer should not exceed 2 hours.
2. Detection by Fluorescence Microscopy
(1) Seeding and culture. Seed cells in multi‑well plates (e.g., 96‑well plates), cell culture dishes, or cell coverslips. Treat cells as required by experimental design.
(2) Washing (optional). For adherent cells, remove culture medium and wash cells once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.
(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution: typically 100 μL per well for 96‑well plates, 250 μL for 24‑well plates, 500 μL for 12‑well plates, and 1 mL for 6‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.
(4) Detection. After incubation, observe staining under a fluorescence microscope (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).
3. Detection by Flow Cytometry
(1) Cell preparation. Harvest adherent cells by trypsin digestion, resuspend in medium, and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Recommended cell amount per sample is 10⁶ cells.
(2) Staining. To the pellet of 10⁶ cells from the previous step, add 1 mL Fluo‑3 Staining Solution and resuspend into a single‑cell suspension. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.
Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.
Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.
(3) Detection. After incubation, analyze directly by flow cytometry, or centrifuge at 250–1000×g for 5 min at room temperature to pellet cells, remove liquid, resuspend in 0.5 mL Assay Buffer per sample, and analyze (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).
Note 1: Use unstained cells in Assay Buffer as the negative control for flow cytometry setup.
Note 2: As flow cytometry is highly sensitive, the probe concentration used may be lower than for microscopy; adjust the dilution of Fluo‑3 AM according to cell type and actual staining.
4. Detection by Fluorescence Microplate Reader
(1) Seeding and culture. Seed cells in black 96‑well plates. Cell number per well should be controlled within 100–10,000, typically 2000–5000. Treat cells as required by experimental design.
(2) Washing (optional). For adherent cells, remove culture medium and wash once with PBS. For suspension cells, centrifuge at 250–1000×g for 5 min at room temperature, discard supernatant, and wash once with PBS. Phenol red or serum may interfere with detection; a vacuum pump is recommended for removing medium and PBS. Washing may be omitted if residual liquid can be thoroughly removed. One wash generally reduces background fluorescence, but good staining can be achieved without washing.
(3) Staining. Add an appropriate volume of Fluo‑3 Staining Solution, typically 100 μL per well in 96‑well plates. Incubate at 37 ℃ for 30 min in the dark. Incubation time can be adjusted between 10 and 60 min.Note 1: For initial experiments with uncertain temperature and time, test incubation at 37 ℃ for 30 min first. Shorten time or lower temperature if excessive cell death occurs; extend time if fluorescence intensity is too weak.Note 2: Cells may be washed 1–3 times with PBS or HBSS after incubation.
(4) Detection. After incubation, measure using a fluorescence microplate reader (Fluo‑3 AM emits green fluorescence, Ex/Em = 490/525 nm).The effect of drug stimulation can be determined by comparing RFU (Relative Fluorescence Units) between control and treated groups.
References
1. Clapham DE. Cell. 2007. 131(6):1047–58.
2. Wu N, Nishioka WK, Derecki NC, Maher MP. Sci Rep. 2019. 9(1):12692.
3. Hagen BM, Boyman L, Kao JP, Lederer WJ. Cell Calcium. 2012. 52(2):170–81.
规格
英文别名
Fluo-3 Calcium Assay Kit
规格或纯度
BioReagent, 生物染色剂, 适用于荧光分析, 用于显微镜
英文名称
Fluo-3 Calcium Ion Detection Kit
应用
Fluorescence Analysis, 细胞染色, 钙离子检测
敏感性
Light-sensitive
Emission(nm)
525nm
Excitation(nm)
Blue Laser(488nm)
Ex/Em(nm)
Ex:490nm;Em:525nm
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
各组分在相应的储存条件下保质期1年。
组分与储存
| F1520956 | 物理外观 | 组件 | 200 T | 储存温度 |
| F1520956A | 液体 | Fluo-3 AM (500×) | 40 μL | -20℃，避光 |
| F1520956B | 液体 | 增溶剂 (500×) | 40 μL | -20℃，避光 |
| F1520956C | 液体 | 检测缓冲液 | 50 mL | -20℃，避光 |
| F1520956D | 液体 | 染色增强剂 (100×) | 200 μL | -20℃，避光 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
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分析证书 | F1520956 |
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溶液计算器
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