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.
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.
复制标题
DOI：
10.1016/j.neuro.2011.01.002
复制
发表时间：
2011-10
期刊：
影响因子：
3.4
通讯作者：
Bowman AB
中科院分区：
文献类型：
--
作者：
Kwakye GF;Li D;Bowman AB
尽管锰 (Mn) 作为多种生理过程必需的微量元素至关重要，但过量的锰会在大脑中积聚，并与基底神经节功能障碍和退化有关。尽管测量锰水平的传统方法具有高灵敏度、有限的化学干扰和多元素优势，但它们缺乏以高通量方式评估锰传输动态的可行性。我们的实验室之前曾报道，亨廷顿病突变纹状体细胞系模型（STHdhQ111/Q111）相对于野生型，在锰暴露后净锰积累减少。为了评估这些纹状体细胞系中锰的转运动力学，我们开发了一种高通量荧光淬灭提取测定（细胞 Fura-2 锰提取测定 - CFMEA）。 CFMEA 利用 360 nm（Ca2+ 等吸光点）激发和 535 nm 发射时 fura-2 荧光的变化，作为细胞总锰含量的间接测量。在此，我们报告CFMEA的建立、发展和应用。具体来说，我们评估了从培养细胞中有效提取和定量检测细胞锰所需的关键提取和测定条件（例如提取缓冲液、温度和 fura-2 浓度）。 Mn 浓度可以通过基于饱和位点特异性结合动力学的标准曲线从 fura-2 荧光猝灭中得出。重要的是，我们表明提取的钙和镁浓度低于 10 μM 对 fura-2 测量 Mn 的影响可以忽略不计。 CFMEA 能够准确测量从培养的纹状体细胞中提取的 Mn 水平，范围至少为 0.1 μM – 10 μM。我们使用两种独立的锰补充方法来验证 CFMEA 在 0 μM – 200 μM 细胞锰暴露范围内的定量准确性。最后，我们利用 CFMEA 实验证实了突变型 HD 纹状体细胞系与野生型细胞相比净 Mn 积累的缺陷。总之，我们开发并应用了一种新的测定方法来评估培养的纹状体细胞系中的锰转运动力学。 CFMEA 提供了一种快速评估细胞毒性和疾病模型中锰转运动力学的方法。
In spite of the essentiality of manganese (Mn) as a trace element necessary for a variety of physiological processes, Mn in excess accumulates in the brain and has been associated with dysfunction and degeneration of the basal ganglia. Despite the high sensitivity, limited chemical interference, and multi-elemental advantages of traditional methods for measuring Mn levels, they lack the feasibility to assess Mn transport dynamics in a high-throughput manner. Our lab has previously reported decreased net Mn accumulation in a mutant striatal cell line model of Huntington’s disease (STHdhQ111/Q111) relative to wild-type following Mn exposure. To evaluate Mn transport dynamics in these striatal cell lines, we have developed a high-throughput fluorescence-quenching extraction assay (Cellular Fura-2 Manganese Extraction Assay - CFMEA). CFMEA utilizes changes in fura-2 fluorescence upon excitation at 360 nm (Ca2+ isosbestic point) and emission at 535 nm, as an indirect measurement of total cellular Mn content. Here, we report the establishment, development, and application of CFMEA. Specifically, we evaluate critical extraction and assay conditions (e.g. extraction buffer, temperature, and fura-2 concentration) required for efficient extraction and quantitative detection of cellular Mn from cultured cells. Mn concentrations can be derived from quenching of fura-2 fluorescence with standard curves based on saturation one-site specific binding kinetics. Importantly, we show that extracted calcium and magnesium concentrations below 10 μM have negligible influence on measurements of Mn by fura-2. CFMEA is able to accurately measure extracted Mn levels from cultured striatal cells over a range of at least 0.1 μM – 10 μM. We have used two independent Mn supplementation approaches to validate the quantitative accuracy of CFMEA over a 0 μM – 200 μM cellular Mn-exposure range. Finally, we have utilized CFMEA to experimentally confirm a deficit in net Mn accumulation in the mutant HD striatal cell line versus wild-type cells. To conclude, we have developed and applied a novel assay to assess Mn transport dynamics in cultured striatal cell lines. CFMEA provides a rapid means of evaluating Mn transport kinetics in cellular toxicity and disease models.
登录
查看更多内容
DOI：
10.1196/annals.1306.009
发表时间：
2004-01-01
期刊：
REDOX-ACTIVE METALS IN NEUROLOGICAL DISORDERS
影响因子：
--
作者：
Dobson, AW;Erikson, KM;Aschner, M
通讯作者：
Aschner, M
影响因子：
5.3
作者：
Oliveira, Jorge M. A.;Chen, Sylvia;Rego, A. Cristina
通讯作者：
Rego, A. Cristina
影响因子：
20.3
作者：
Forbes, JR;Gros, P
通讯作者：
Gros, P
影响因子：
16.2
作者：
Tang, TS;Tu, HP;Bezprozvanny, I
通讯作者：
Bezprozvanny, I
影响因子：
3.5
作者：
Trettel, F;Rigamonti, D;MacDonald, ME
通讯作者：
MacDonald, ME