Fluo-3AM
(Synonyms: 钙荧光探针Fluo3-AM,Fluo-3-pentaacetoxymethyl ester) 目录号 : GC33437
Fluo-3AM是最常用的检测细胞内钙离子浓度的荧光探针之一。
Cas No.:121714-22-5
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1.制备染色液
(1)配置储存液: 在高质量无水DMSO中制备浓度为1-10mM的AM酯储存液；
注意:
① 未使用的储存液分装后在-20℃或-80°C避光保存，避免反复冻融；
② 乙酰氧基甲基酯(AM)易吸潮，冰箱取出后请在干燥的环境放至室温后再开封。开封前请将其短暂离心，以保证粉末落入管底。
(2)配置工作液:用合适的缓冲液(如:无血清和酚红的培养基或PBS)稀释储存液，配制浓度为1-10μM的工作液。
注意:
① AM酯染色工作液制备时，有时需要往储存液中加入适量的20% Pluronic F-127溶液，以增强AM探针的水溶性；
② Pluronic F-127可以防止AM探针在溶液中聚合并促使探针更好进入细胞。但PluronicF-127可降低AM探针的稳定性，因此只建议在配制工作液时加入，不建议加入储存液长期保存；
③ 配制20%(w/v) Pluronic F-127母液:称取100mg Pluronic F-127粉末(货号: GB30090)，加入500μl DMSO，40-50℃加热20-30min，室温保存。如有结晶析出可重新加热溶解，不影响使用；
④ （可选）GLPBIO提供溶解好的Pluronic F-127(20% Solution in DMSO) ，货号：GB30091；
⑤ 添加等体积20% Pluronic F-127溶液到储存液中，从而使Pluronic F-127的最终工作浓度约为0.02%；
⑥ 请根据实际情况调整工作液浓度，现用现配，避免反复冻融。
2.细胞悬浮染色
(1)悬浮细胞：经4°C、1000g离心3-5分钟，弃去上清液，使用PBS或其他缓冲液清洗两次，每次5分钟；
(2)贴壁细胞：使用PBS或其他缓冲液清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min；
(3)加入染料工作溶液重悬细胞，室温或低于室温条件下避光孵育20min-2h。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索；
注：
① AM酯类染料在大部分细胞中的推荐工作浓度为4-5μM，具体使用浓度需根据实验要求进行优化。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尽量使用最低探针浓度；
② （可选）如果细胞内含有机阴离子转运体，可能需要在细胞培养基中加入丙磺舒(GC16825，Probenecid，1-2.5mM)或磺吡酮(GC11049 ，Sulfinpyrazone，0.1-0.25mM)，以降低去酯化 探针的泄露水平。丙磺舒或磺吡酮储存液偏碱，因此加入培养基后需要重新调整pH；
③ 若使用含血清的培养基，血清内酯酶会降解AM，从而降低染料加载效果；而含酚红培养基会使本底值略偏高，建议加入染色工作液前，对细胞清洗2~3次；
④ 降低探针加载温度可能会降低探针的区室化现象。
(4)孵育结束后，经1000g离心5分钟，去除染色液，加入PBS或其他缓冲液清洗2-3次，去除残留探针；
(5)室温再孵育30min以保证细胞内AM的完全去酯化。
3.细胞贴壁染色
(1)在无菌盖玻片上培养贴壁细胞；
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中；
(3)从盖玻片的一角加入100μL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞；
(4) 室温或低于室温条件下避光孵育20min-2h。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索；
(5)孵育结束后吸弃染料工作液，使用PBS或其他缓冲液清洗盖玻片2~3次；
(6)室温孵育30min。
4.显微镜检测：Fluo-3AM的最大激发/发射波长为488/526nm。
注意事项：
1）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。
2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
Fluo-3AM is one of the most commonly used fluorescent probes to detect intracellular calcium ion concentration.After it penetrates the cell membrane and enters the cell, it is cleaved by the esterase in the cell to form Fluo-3, so as to be retained in the cell.Fluo-3 is almost non-fluorescent when it exists in the form of free ligand, but it can produce strong fluorescence when it combines with intracellular calcium ions, with a maximum excitation wavelength of 506nm and a maximum emission wavelength of 526nm. In actual detection, the recommended excitation wavelength is about 488nm and the emission wavelength is 525-530nm. Changes in intracellular calcium concentration can be detected using laser confocal microscopy or flow cytometry[1-4].
References:
[1]. Loughrey CM, MacEachern KE, et,al. Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics. Cell Calcium. 2003 Jul;34(1):1-9. doi: 10.1016/s0143-4160(03)00012-5. PMID: 12767887.
[2]. Ji S, Li S, Zhao X, et,al.Protective role of phenylethanoid glycosides, Torenoside B and Savatiside A, in Alzheimer's disease. Exp Ther Med. 2019 May;17(5):3755-3767. doi: 10.3892/etm.2019.7355. Epub 2019 Mar 7. PMID: 30988761; PMCID: PMC6447766.
[3]. Williams PDE, Verma S, et,al.Adapting techniques for calcium imaging in muscles of adult Brugia malayi. Invert Neurosci. 2020 Aug 16;20(3):12. doi: 10.1007/s10158-020-00247-1. PMID: 32803437; PMCID: PMC7891862.
[4]. Williams PDE, Kashyap SS, et,al.Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine. Sci Rep. 2022 Dec 9;12(1):21317. doi: 10.1038/s41598-022-25648-7. PMID: 36494409; PMCID: PMC9734116.
Fluo-3AM是最常用的检测细胞内钙离子浓度的荧光探针之一。它穿透细胞膜进入细胞后被细胞内的酯酶剪切形成 Fluo-3 ,从而被滞留在细胞内。 Fluo-3 若以游离配体形式存在时几乎是非荧光性的,但是当它与细胞内钙离子结合后可以产生较强的荧光,最大激发波长为 506nm,最大发射波长为 526nm 实际检测时推荐使用的激发波长为 488nm 左右,发射波长为 525~530nm。 可以使用激光共聚焦显微镜或流式细胞仪检测细胞内钙离子浓度的变化[1-4]。
| Cas No. | 121714-22-5 | SDF | |
| 别名 | 钙荧光探针Fluo3-AM,Fluo-3-pentaacetoxymethyl ester | ||
| Canonical SMILES | ClC(C=C1C(C2=CC(OCCOC(C=C(C)C=C3)=C3N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)=C(N(CC(OCOC(C)=O)=O)CC(OCOC(C)=O)=O)C=C2)=C(C=C4Cl)C5=CC4=O)=C(C=C1O5)OCOC(C)=O | ||
| 分子式 | C51H50Cl2N2O23 | 分子量 | 1129.85 |
| 溶解度 | Acetonitrile: 2 mg/ml,DMSO: 0.9 mg/ml | 储存条件 | Store at -20°C,protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 0.8851 mL | 4.4254 mL | 8.8507 mL |
| 5 mM | 0.177 mL | 0.8851 mL | 1.7701 mL |
| 10 mM | 0.0885 mL | 0.4425 mL | 0.8851 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
The involvement of PI3K-mediated and L-VGCC-gated transient Ca2+ influx in 17β-estradiol-mediated protection of retinal cells from H2O2-induced apoptosis with Ca2+ overload
PLoS One 2013 Nov 5;8(11):e77218.PMID:24223708DOI:10.1371/journal.pone.0077218.
Intracellular calcium concentration ([Ca(2+)]i) plays an important role in regulating most cellular processes, including apoptosis and survival, but its alterations are different and complicated under diverse conditions. In this study, we focused on the [Ca(2+)]i and its control mechanisms in process of hydrogen peroxide (H2O2)-induced apoptosis of primary cultured Sprague-Dawley (SD) rat retinal cells and 17β-estradiol (βE2) anti-apoptosis. Fluo-3AM was used as a Ca(2+) indicator to detect [Ca(2+)]i through fluorescence-activated cell sorting (FACS), cell viability was assayed using MTT assay, and apoptosis was marked by Hoechst 33342 and annexin V/Propidium Iodide staining. Besides, PI3K activity was detected by Western blotting. Results showed: a) 100 μM H2O2-induced retinal cell apoptosis occurred at 4 h after H2O2 stress and increased in a time-dependent manner, but [Ca(2+)]i increased earlier at 2 h, sustained to 12 h, and then recovered at 24 h after H2O2 stress; b) 10 μM βE2 treatment for 0.5-24 hrs increased cell viability by transiently increasing [Ca(2+)]i, which appeared only at 0.5 h after βE2 application; c) increased [Ca(2+)]i under 100 µM H2O2 treatment for 2 hrs or 10 µM βE2 treatment for 0.5 hrs was, at least partly, due to extracellular Ca(2+) stores; d) importantly, the transiently increased [Ca(2+)]i induced by 10 µM βE2 treatment for 0.5 hrs was mediated by the phosphatidylinositol-3-kinase (PI3K) and gated by the L-type voltage-gated Ca(2+) channels (L-VGCC), but the increased [Ca(2+)]i induced by 100 µM H2O2 treatment for 2 hrs was not affected; and e) pretreatment with 10 µM βE2 for 0.5 hrs effectively protected retinal cells from apoptosis induced by 100 µM H2O2, which was also associated with its transient [Ca(2+)]i increase through L-VGCC and PI3K pathway. These findings will lead to better understanding of the mechanisms of βE2-mediated retinal protection and to exploration of the novel therapeutic strategies for retina degeneration.
635 nm LED irradiation may prevent endoplasmic reticulum stress in MC3T3-E1 cells
J Mol Histol 2022 Feb;53(1):75-83.PMID:34676487DOI:10.1007/s10735-021-10034-w.
Although endoplasmic reticulum (ER) stress is thought to be involved in various diseases such as cancer, metabolic, and inflammatory disorders, the relationship between ER stress and bone diseases, are remains unclear. Tunicamycin-treated MC3T3-E1 osteoblasts were used as the ER stress model in this study. 635 nm light-emitting diode irradiation (635 nm-IR) was carried out for 1 h before and after inducing ER stress. To investigate the effects of 635 nm-IR on ER stress-induced MC3T3-E1 osteoblasts and the underlying mechanism, western blot, reverse transcription polymerase chain reaction, alkaline phosphatase and Alizarin red staining, 2',7'-dichlorodyhydrofluorescein diacetate assay, Fluo-3AM and immunocytochemistry were performed. Pretreatment with 635 nm-IR effectively prevented intracellular reactive oxygen species production and alleviated ER stress through the pancreatic ER kinase (PERK)-eukaryotic initiation factor 2 (eIF2)-activating transcription factor 4 (ATF4)-nuclear factor-like 2 (Nrf2) signaling pathway. Hence, 635 nm-IR may serve a protective role in the treatment of ER stress-related bone diseases.
Diethylcarbamazine, TRP channels and Ca2+ signaling in cells of the Ascaris intestine
Sci Rep 2022 Dec 9;12(1):21317.PMID:36494409DOI:10.1038/s41598-022-25648-7.
The nematode parasite intestine absorbs nutrients, is involved in innate immunity, can metabolize xenobiotics and as we show here, is also a site of action of the anthelmintic, diethylcarbamazine. Diethylcarbamazine (DEC) is used to treat lymphatic filariasis and activates TRP-2, GON-2 & CED-11 TRP channels in Brugia malayi muscle cells producing spastic paralysis. DEC also has stimulatory effects on ascarid nematode parasites. Using PCR techniques, we detected, in Ascaris suum intestine, message for: Asu-trp-2, Asu-gon-2, Asu-ced-11, Asu-ocr-1, Asu-osm-9 and Asu-trpa-1. Comparison of amino-acid sequences of the TRP channels of B. malayi, and A. suum revealed noteworthy similarity, suggesting that the intestine of Ascaris will also be sensitive to DEC. We used Fluo-3AM as a Ca2+ indicator and observed characteristic unsteady time-dependent increases in the Ca2+ signal in the intestine in response to DEC. Application of La3+ and the TRP channel inhibitors, 2-APB or SKF 96365, inhibited DEC mediated increases in intracellular Ca2+. These observations are important because they emphasize that the nematode intestine, in addition to muscle, is a site of action of DEC as well as other anthelmintics. DEC may also enhance the Ca2+ toxicity effects of other anthelmintics acting on the intestine or, increase the effects of other anthelmintics that are metabolized and excreted by the nematode intestine.
Stimulation of platelet apoptosis by balhimycin
Biochem Biophys Res Commun 2013 May 31;435(2):323-6.PMID:23399563DOI:10.1016/j.bbrc.2013.01.120.
Glycopeptides, such as vancomycin, are powerful antibiotics against methicillin-resistant Staphylococcus aureus. Balhimycin, a glycopeptide antibiotic isolated from Amycolatopsis balhimycina, is similarly effective as vancomycin. Side effects of vancomycin include triggering of platelet apoptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulation of apoptosis may involve increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization and/or caspase activation. An effect of balhimycin on apoptosis has, however, never been reported. The present study thus tested whether balhimycin triggers platelet apoptosis. Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V-binding, cytosolic Ca(2+) activity from Fluo-3AM fluorescence, ceramide formation utilizing antibodies, mitochondrial potential from DiOC6 fluorescence, and caspase-3 activity utilizing antibodies. As a result, a 30 min exposure to balhimycin significantly decreased cell volume (≥1 μg/ml), triggered annexin V binding (≥1 μg/ml), increased cytosolic Ca(2+) activity (≥1 μg/ml), stimulated ceramide formation (≥10 μg/ml), depolarized mitochondria (≥1 μg/ml) and activated caspase-3 (≥1 μg/ml). Cell membrane scrambling and caspase-3 activation were virtually abrogated by removal of extracellular Ca(2+). Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca(2+), but not on activation of caspases.
Adapting techniques for calcium imaging in muscles of adult Brugia malayi
Invert Neurosci 2020 Aug 16;20(3):12.PMID:32803437DOI:10.1007/s10158-020-00247-1.
Brugia malayi is a human filarial nematode parasite that causes lymphatic filariasis or 'elephantiasis' a disfiguring neglected tropical disease. This parasite is a more tractable nematode parasite for the experimental study of anthelmintic drugs and has been studied with patch-clamp and RNAi techniques. Unlike in C. elegans however, calcium signaling in B. malayi or other nematode parasites has not been achieved, limiting the studies of the mode of action of anthelmintic drugs. We describe here the development of calcium imaging methods that allow us to characterize changes in cellular calcium in the muscles of B. malayi. This is a powerful technique that can help in elucidating the mode of action of selected anthelmintics. We developed two approaches that allow the recording of calcium signals in the muscles of adult B. malayi: (a) soaking the muscles with Fluo-3AM, promoting large-scale imaging of multiple cells simultaneously and, (b) direct insertion of Fluo-3 using microinjection, providing the possibility of performing dual calcium and electrophysiological recordings. Here, we describe the techniques used to optimize dye entry into the muscle cells and demonstrate that detectable increases in Fluo-3 fluorescence to elevated calcium concentrations can be achieved in B. malayi using both techniques.