Kwakye Gunnar F, Li Daphne, Kabobel Olympia A, Bowman Aaron B
Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Curr Protoc Toxicol. 2011 May;Chapter 12:Unit12.18. doi: 10.1002/0471140856.tx1218s48.
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.
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。
