BCECF-AM
(Synonyms: 2',7'-二-(2-羧乙基)-5(6)-羧基荧光素乙酰甲酯,BCECF Acetoxymethyl ester) 目录号 : GC14603A fluorescent indicator for estimating intracellular pH
Cas No.:117464-70-7
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
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Cell experiment: |
PASMCs are placed in a laminar flow cell chamber perfused with HBSS with pH adjusted to 7.4. pHi is measured in cells incubated with the membrane permeant (acetoxymethyl ester) form of the pH-sensitive fluorescent dye BCECF-AM for 60 min at 37°C under an atmosphere of 20% O2-5% CO2. Cells are then washed with HBSS for 15 min at 37°C to remove extracellular dye and allow complete de-esterification of cytosolic dye. Ratiometric measurement of BCECF fluorescence is performed on a workstation consisting of a Nikon TSE 100 Ellipse inverted microscope with epi-fluorescence attachments. The light beam from a xenon arc lamp is filtered by interference filters at 490 and 440 nm, and focused onto the PASMCS under examination via a 20× fluorescence objective. Light emitted from the cell at 530 nm is returned through the objective and detected by an imaging camera. An electronic shutter is used to minimize photobleaching of dye. Protocols are executed and data collected on-line with InCyte software. pHi is estimated from in situcalibration after each experiment. Cells are perfused with a solution containing (in mM): 105 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES-Tris and 0.01 nigericin to allow pHi to equilibrate to external pH. A two point calibration is created from fluorescence measured as pHi is adjusted with KOH from 6.5 to 7.5. Intracellular H+ ion concentration ([H+]i) is determined from pHi using the formula: pHi = −log ([H+]i). |
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References: [1]. Clark Undem, et al. Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase. PLoS One. 2012; 7(9): e46303. |
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BCECF AM ester is a widely-used fluorescent indicator for estimating intracellular pH.[1],[2] This membrane-permeant compound is modified by intracellular esterases to produce BCECF, a polar fluorescein derivative that is retained by cells. Intracellular BCECF is stable and has an efflux half-life of greater than two hours.[3] Intracellular pH measurements are made by ratio imaging, which involves determining the pH-dependent ratio of emission intensity (detected at 535 nm) when the dye is excited at 490 nm vs. the emission intensity when excited at 440 nm.[2],[4] This approach is amenable to either spectrofluorometry or flow cytometry.[2],[5] BCECF AM can also be used to investigate intracellular changes in other ions, including potassium.[6]
Reference:
[1]. Rink, T.J., Tsien, R.Y., and Pozzan, T. Cytoplasmic pH and free Mg2+ in lymphocytes. Journal of Cell Biology 95(1), 189-196 (1982).
[2]. Ozkan, P., and Mutharasan, R. A rapid method for measuring intracellular pH using BCECF-AM. Biochimica et Biophysica Acta 1572(1), 143-148 (2002).
[3]. Dive, C., Cox, H., Watson, J.V., et al. Polar fluorescein derivatives as improved substrate probes for flow cytoenzymological assay of cellular esterases. Mol.Cell Probe 2(2), 131-145 (1988).
[4]. O'Connor, N., and Silver, R.B. Ratio imaging: Practical considerations for measuring intracellular Ca2+ and pH in living cells. Methods Cell Biol. 81, 415-433 (2007).
[5]. Chow, S., Hedley, D., and Tannock, I. Flow cytometric calibration of intracellular pH measurements in viable cells using mixtures of weak acids and bases. Cytometry 24, 360-367 (1996).
[6]. Balkay, L., Márián, T., Emri, M., et al. Flow cytometric determination of intracellular free potassium concentration. Cytometry 28, 42-49 (1997).
| Cas No. | 117464-70-7 | SDF | |
| 别名 | 2',7'-二-(2-羧乙基)-5(6)-羧基荧光素乙酰甲酯,BCECF Acetoxymethyl ester | ||
| 化学名 | 3',6'-bis[(acetyloxy)methoxy]-5(or 6)-[[(acetyloxy)methoxy]carbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-2',7'-dipropanoic acid, 2',7'-bis[(acetyloxy)methyl] ester | ||
| Canonical SMILES | O=C(C)OCOC1=C(CCC(OCOC(C)=O)=O)C=C(C2(C(C=CC(C(OCOC(C)=O)=O)=C3)=C3C(O2)=O)C(C(O4)=C5)=CC(CCC(OCOC(C)=O)=O)=C5OCOC(C)=O)C4=C1.O=C(C)OCOC6=C(CCC(OCOC(C)=O)=O)C=C(C7(C(C=C(C(OCOC(C)=O)=O)C=C8)=C8C(O7)=O)C(C(O9)=C%10)=CC(CCC(OCOC(C)=O)=O)=C%10OCOC(C)=O)C9=C6 | ||
| 分子式 | C42H40O21 | 分子量 | 880.7 |
| 溶解度 | Soluble in ,Acetonitrile, DMF, DMSO | 储存条件 | Store at -20°C, protect from light,unstable in solution, ready to use. |
| 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 | 1.1355 mL | 5.6773 mL | 11.3546 mL |
| 5 mM | 0.2271 mL | 1.1355 mL | 2.2709 mL |
| 10 mM | 0.1135 mL | 0.5677 mL | 1.1355 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。