Glutaraldehyde - A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells
Glutaraldehyde - A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells
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DOI：
10.3389/fphys.2019.00514
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发表时间：
2019-05-14
影响因子：
4
通讯作者：
Kaestner, Lars
中科院分区：
文献类型：
--
作者：
Abay, Asena;Simionato, Greta;Kaestner, Lars
戊二醛是生物医学研究中用于固定细胞的众所周知的物质。由于溶血性贫血通常与红细胞形状改变偏离双凹面盘形状有关，因此通常保存这些形状用于成像，特别是3D成像，如共聚焦显微镜，扫描电子显微镜或扫描探针显微镜是一种常见的愿望。沿着的是细胞硬度的增加。在红细胞的背景下，这种增加的刚性通常用于模拟疟疾感染的红细胞，因为它们也比健康的红细胞更硬。然而，戊二醛的使用与许多缺陷相关：（i）虽然通过应用增加浓度的戊二醛来增加刚度是一个模拟过程，但固定是一个相当数字化的事件（全部或无）;（ii）戊二醛的添加以浓度依赖性方式极大地改变了渗透压，因此细胞形状可能会变形;（iii）戊二醛批次在它们的性质上不同，特别是在单体和聚合物的比例上不同;（iv）处理缺陷，如诱导需要考虑的红细胞形状或细胞密度变化的剪切伪影，例如，当处理流动中的细胞时;（v）与活细胞相比，戊二醛处理的红细胞需要不同的染色方法，例如，因为戊二醛本身诱导强荧光。在本文中，我们提供了关于戊二醛对健康和病理性红细胞的微妙使用以及如何处理或规避陷阱的文件。
Glutaraldehyde is a well-known substance used in biomedical research to fix cells. Since hemolytic anemias are often associated with red blood cell shape changes deviating from the biconcave disk shape, conservation of these shapes for imaging in general and 3D-imaging in particular, like confocal microscopy, scanning electron microscopy or scanning probe microscopy is a common desire. Along with the fixation comes an increase in the stiffness of the cells. In the context of red blood cells this increased rigidity is often used to mimic malaria infected red blood cells because they are also stiffer than healthy red blood cells. However, the use of glutaraldehyde is associated with numerous pitfalls: (i) while the increase in rigidity by an application of increasing concentrations of glutaraldehyde is an analog process, the fixation is a rather digital event (all or none); (ii) addition of glutaraldehyde massively changes osmolality in a concentration dependent manner and hence cell shapes can be distorted; (iii) glutaraldehyde batches differ in their properties especially in the ratio of monomers and polymers; (iv) handling pitfalls, like inducing shear artifacts of red blood cell shapes or cell density changes that needs to be considered, e.g., when working with cells in flow; (v) staining glutaraldehyde treated red blood cells need different approaches compared to living cells, for instance, because glutaraldehyde itself induces a strong fluorescence. Within this paper we provide documentation about the subtle use of glutaraldehyde on healthy and pathologic red blood cells and how to deal with or circumvent pitfalls.