细胞呋喃-2 锰提取测定法（CFMEA）。
Cellular fura-2 manganese extraction assay (CFMEA).
作者信息
Kwakye Gunnar F, Li Daphne, Kabobel Olympia A, Bowman Aaron B
机构信息
Vanderbilt University Medical Center, Nashville, Tennessee, USA.
出版信息
Curr Protoc Toxicol. 2011 May;Chapter 12:Unit12.18. doi: 10.1002/0471140856.tx1218s48.
Abstract
Cellular manganese (Mn) uptake and transport dynamics can be measured using a cellular fura-2 manganese extraction assay (CFMEA). The assay described here uses immortalized murine striatal cell line and primary cortical astrocytes, but the method is equally adaptable to other cultured mammalian cells. An ultrasensitive fluorescent nucleic acid stain for quantification of double-stranded DNA (dsDNA) in solution, Quant-iT PicoGreen, has been utilized for normalization of Mn concentration in the cultured cells, following Mn (II) chloride (MnCl(2)) exposure. Depending on the cell type and density, other methods, e.g., protein determination assays or cell counts, may also be used for normalization. Methods are described for rapidly stopping Mn uptake and transport processes at specified times, extraction, and quantification of cellular Mn content, and normalization of Mn levels to dsDNA concentration.
摘要
细胞锰（Mn）摄取和转移动力学可以使用细胞fura-2锰提取测定法（CFMEA）来测量。这里描述的测定法使用永生化小鼠纹状体细胞系和原代皮质星形胶质细胞，但该方法同样适用于其他培养的哺乳动物细胞。一种用于定量溶液中双链DNA（dsDNA）的超灵敏荧光核酸染料Quant-iT PicoGreen，已被用于在氯化锰（MnCl₂）暴露后对培养细胞中的锰浓度进行标准化。根据细胞类型和密度，也可以使用其他方法，例如蛋白质测定法或细胞计数，进行标准化。文中描述了在特定时间快速停止锰摄取和转运过程、提取和定量细胞锰含量以及将锰水平标准化为dsDNA浓度的方法。
相似文献
1
Cellular fura-2 manganese extraction assay (CFMEA).细胞呋喃-2 锰提取测定法（CFMEA）。
Curr Protoc Toxicol. 2011 May;Chapter 12:Unit12.18. doi: 10.1002/0471140856.tx1218s48.
2
Novel high-throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation.新型高通量检测方法评估亨廷顿病纹状体细胞模型中的细胞内锰水平，证实了锰积累缺陷。
Neurotoxicology. 2011 Oct;32(5):630-9. doi: 10.1016/j.neuro.2011.01.002. Epub 2011 Jan 14.
3
Optimization of fluorescence assay of cellular manganese status for high throughput screening.优化细胞锰状态荧光检测法用于高通量筛选。
J Biochem Mol Toxicol. 2013 Jan;27(1):42-9. doi: 10.1002/jbt.21457. Epub 2012 Nov 20.
4
Phosphatidylinositol 3 kinase (PI3K) modulates manganese homeostasis and manganese-induced cell signaling in a murine striatal cell line.磷脂酰肌醇 3 激酶（PI3K）调节锰稳态和锰诱导的细胞信号在一个小鼠纹状体细胞系。
Neurotoxicology. 2018 Jan;64:185-194. doi: 10.1016/j.neuro.2017.07.026. Epub 2017 Aug 2.
5
The uptake of manganese in brain endothelial cultures.
Neurotoxicology. 2002 Jul;23(2):165-8. doi: 10.1016/s0161-813x(02)00056-6.
6
Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese.鉴定一种选择性锰离子载体，可实现细胞锰的非致死定量。
J Biol Chem. 2020 Mar 20;295(12):3875-3890. doi: 10.1074/jbc.RA119.009781. Epub 2020 Feb 11.
7
Direct CSF injection of MnCl(2) for dynamic manganese-enhanced MRI.经脑脊液直接注射氯化锰用于动态锰增强磁共振成像
Magn Reson Med. 2004 May;51(5):978-87. doi: 10.1002/mrm.20047.
8
A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.一种使用荧光染料fura-2测定无机多磷酸盐的新方法。
Anal Biochem. 1997 Mar 15;246(2):176-84. doi: 10.1006/abio.1996.9998.
9
Glutamate/aspartate transporter (GLAST), taurine transporter and metallothionein mRNA levels are differentially altered in astrocytes exposed to manganese chloride, manganese phosphate or manganese sulfate.谷氨酸/天冬氨酸转运体（GLAST）、牛磺酸转运体和金属硫蛋白的mRNA水平在暴露于氯化锰、磷酸锰或硫酸锰的星形胶质细胞中发生不同程度的改变。
Neurotoxicology. 2002 Sep;23(3):281-8. doi: 10.1016/s0161-813x(02)00041-4.
10
The protective effects of riluzole on manganese-induced disruption of glutamate transporters and glutamine synthetase in the cultured astrocytes.雷利佐生对锰诱导的培养星形胶质细胞谷氨酸转运体和谷氨酰胺合成酶破坏的保护作用。
Biol Trace Elem Res. 2012 Aug;148(2):242-9. doi: 10.1007/s12011-012-9365-1. Epub 2012 Mar 6.
引用本文的文献
1
IGF/mTORC1/S6 Signaling Is Potentiated and Prolonged by Acute Loading of Subtoxicological Manganese Ion.急性低毒锰离子负荷会增强和延长 IGF/mTORC1/S6 信号通路。
Biomolecules. 2023 Aug 8;13(8):1229. doi: 10.3390/biom13081229.
2
Insights into the regulation of cellular Mn homeostasis via TMEM165.通过 TMEM165 深入了解细胞内锰稳态的调节。
Biochim Biophys Acta Mol Basis Dis. 2023 Aug;1869(6):166717. doi: 10.1016/j.bbadis.2023.166717. Epub 2023 Apr 14.
3
Impaired XK recycling for importing manganese underlies striatal vulnerability in Huntington's disease.纹状体易损性的基础是亨廷顿病中锰导入的 XK 循环受损。
J Cell Biol. 2022 Oct 3;221(10). doi: 10.1083/jcb.202112073. Epub 2022 Sep 13.
4
Identification of Three Small Molecules That Can Selectively Influence Cellular Manganese Levels in a Mouse Striatal Cell Model.鉴定三种可在小鼠纹状体细胞模型中选择性影响细胞内锰水平的小分子。
Molecules. 2021 Feb 22;26(4):1175. doi: 10.3390/molecules26041175.
5
Manganese-induced Mitochondrial Dysfunction Is Not Detectable at Exposures Below the Acute Cytotoxic Threshold in Neuronal Cell Types.锰诱导的线粒体功能障碍在低于神经细胞类型的急性细胞毒性阈值的暴露水平下是不可检测的。
Toxicol Sci. 2020 Aug 1;176(2):446-459. doi: 10.1093/toxsci/kfaa079.
6
Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese.鉴定一种选择性锰离子载体，可实现细胞锰的非致死定量。
J Biol Chem. 2020 Mar 20;295(12):3875-3890. doi: 10.1074/jbc.RA119.009781. Epub 2020 Feb 11.
7
Huntington's disease associated resistance to Mn neurotoxicity is neurodevelopmental stage and neuronal lineage dependent.亨廷顿病相关的锰神经毒性抗性与神经发育阶段和神经元谱系有关。
Neurotoxicology. 2019 Dec;75:148-157. doi: 10.1016/j.neuro.2019.09.007. Epub 2019 Sep 20.
8
Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn is driven by an active Ca-coupled exchange.锌转运蛋白 10（ZnT10）依赖性细胞内锰的外排是由活性钙耦交换驱动的。
J Biol Chem. 2019 Apr 12;294(15):5879-5889. doi: 10.1074/jbc.RA118.006816. Epub 2019 Feb 12.
9
Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca entry.慢性低氧后膜胆固醇减少限制了 Orai1 介导的肺内皮细胞 Ca 内流。
Am J Physiol Heart Circ Physiol. 2018 Feb 1;314(2):H359-H369. doi: 10.1152/ajpheart.00540.2017. Epub 2017 Nov 3.
10
Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity.小胶质细胞在锰神经毒性中增强星形胶质细胞的炎症激活。
J Neuroinflammation. 2017 May 5;14(1):99. doi: 10.1186/s12974-017-0871-0.
本文引用的文献
1
Interactions between excessive manganese exposures and dietary iron-deficiency in neurodegeneration.锰暴露过度与神经退行性变中铁缺乏症的相互作用。
Environ Toxicol Pharmacol. 2005 May;19(3):415-21. doi: 10.1016/j.etap.2004.12.053.
2
Novel high-throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation.新型高通量检测方法评估亨廷顿病纹状体细胞模型中的细胞内锰水平，证实了锰积累缺陷。
Neurotoxicology. 2011 Oct;32(5):630-9. doi: 10.1016/j.neuro.2011.01.002. Epub 2011 Jan 14.
3
Altered manganese homeostasis and manganese toxicity in a Huntington's disease striatal cell model are not explained by defects in the iron transport system.亨廷顿病纹状体细胞模型中锰稳态的改变和锰毒性不能用铁转运系统的缺陷来解释。
Toxicol Sci. 2010 Sep;117(1):169-79. doi: 10.1093/toxsci/kfq174. Epub 2010 Jun 13.
4
Disease-toxicant screen reveals a neuroprotective interaction between Huntington's disease and manganese exposure.疾病-毒物筛选揭示亨廷顿病与锰暴露之间的神经保护相互作用。
J Neurochem. 2010 Jan;112(1):227-37. doi: 10.1111/j.1471-4159.2009.06445.x. Epub 2009 Oct 21.
5
Effect of manganese exposure on intracellular Ca2+ homeostasis and expression of NMDA receptor subunits in primary cultured neurons.锰暴露对原代培养神经元细胞内 Ca2+ 稳态和 NMDA 受体亚单位表达的影响。
Neurotoxicology. 2009 Nov;30(6):941-9. doi: 10.1016/j.neuro.2009.07.011. Epub 2009 Jul 28.
6
Calcium homeostasis and mitochondrial dysfunction in striatal neurons of Huntington disease.亨廷顿病纹状体神经元中的钙稳态与线粒体功能障碍
J Biol Chem. 2008 Feb 29;283(9):5780-9. doi: 10.1074/jbc.M704704200. Epub 2007 Dec 21.
7
Manganese: recent advances in understanding its transport and neurotoxicity.锰：在理解其转运与神经毒性方面的最新进展
Toxicol Appl Pharmacol. 2007 Jun 1;221(2):131-47. doi: 10.1016/j.taap.2007.03.001. Epub 2007 Mar 12.
8
Mitochondrial-dependent Ca2+ handling in Huntington's disease striatal cells: effect of histone deacetylase inhibitors.亨廷顿舞蹈病纹状体细胞中依赖线粒体的钙离子处理：组蛋白去乙酰化酶抑制剂的作用
J Neurosci. 2006 Oct 25;26(43):11174-86. doi: 10.1523/JNEUROSCI.3004-06.2006.
9
Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1.亨廷顿蛋白和亨廷顿蛋白相关蛋白1影响由1,4,5-三磷酸肌醇受体1介导的神经元钙信号传导。
Neuron. 2003 Jul 17;39(2):227-39. doi: 10.1016/s0896-6273(03)00366-0.
10
Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane.铁、锰和钴通过位于质膜上表达的Nramp1（Slc11a1）和Nramp2（Slc11a2）进行转运。
Blood. 2003 Sep 1;102(5):1884-92. doi: 10.1182/blood-2003-02-0425. Epub 2003 May 15.
Zotero 插件