Improving AM ester calcium dye loading efficiency
Improving AM ester calcium dye loading efficiency
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DOI：
10.1016/j.jneumeth.2014.11.010
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发表时间：
2015-01-30
影响因子：
3
通讯作者：
Wahle, Petra
中科院分区：
文献类型：
--
作者：
Hamad, Mohammad I. K.;Krause, Martin;Wahle, Petra
背景：钙成像揭示了神经元功能的钙依赖性机制。乙酰氧基甲酯 (AM) 染料广泛用于钙成像。使用作为表面活性剂的 Pluronic F127 (PF127) 和作为溶剂的二甲基亚砜 (DMSO) 来溶解染料，但浓度因方案而异。这些物质如何影响装载效率尚未得到很好的表征。新方法：我们的目的是表征切片培养物中的染料装载量。我们确定了表面活性剂、溶剂和染料的最低浓度。目前的研究表明，AM 染料加载的效率取决于 PF127 的初始库存浓度。降低 PF127 和 DMSO 浓度可以提高上样效率。结果：成功上样染料需要 Pluronic 和 DMSO。然而，将染料溶解在较低浓度的 PF127 中可产生更好的染色效率。此外，将 DMSO 浓度降低至 0.25% 左右可提高效率。该策略允许使用标准双光子或共焦显微镜监测神经元活动。标记的细胞显示自发的和诱发的钙瞬变，加载后长达 24 小时的重复测量表明该方法对神经元功能不会有害。与现有方法比较：将 AM 染料溶解在较低浓度的 PF127 中，结合了高加载效率的优点，保留了细胞活力和功能完整性，并允许在数小时和数天内重复测量。此外，我们发现染料本身可以稀释至1μM的终浓度，从而降低了实验成本。结论：该方法是切片培养物中钙成像的最佳方法。 (C) 2014 Elsevier B.V. 保留所有权利。
Background: Calcium imaging has unraveled the calcium-dependent mechanisms underlying neuronal function. Acetoxymethyl ester (AM) dyes are widely employed for calcium imaging. Pluronic F127 (PF127) as a surfactant and dimethyl sulfoxide (DMSO) as a solvent are used to dissolve the dyes, but concentrations vary between protocols. How these substances affect loading efficiency is not well characterized.New method: We aimed to characterize dye loading in slice cultures. We determined minimum concentrations of surfactant, solvent and dye. The current study shows that the efficiency of AM dye loading depends on the initial stock concentration of PF127. Lowering the PF127 and DMSO concentrations can improve the loading efficiency.Results: Both, pluronic and DMSO are required for successful dye loading. However, dissolving the dyes in lower concentrations of PF127 yielded better staining efficiency. Moreover, lowering the DMSO concentration to similar to 0.25% improves the efficiency. The strategy allows standard two-photon or confocal microscope monitoring of neuronal activity. The labeled cells display spontaneous and evoked calcium transients, and repetitive measurements for up to 24 h after loading indicate that the method is not deleterious to neuronal function.Comparison with existing method(s): Dissolving the AM dyes in lower concentrations of PF127 combines the advantages of high loading efficiency, preserves cell viability and functional integrity, and allows repetitive measures over hours and days. Moreover, we found that the dye itself can be diluted to a final concentration of 1 mu M which reduces the experimental costs.Conclusion: The method is optimal for calcium imaging in slice cultures. (C) 2014 Elsevier B.V. All rights reserved.