Abstract：
Broadly speaking, cell tracking dyes are fluorescent compounds that bind stably to components on or within the cells so the fate of the labeled cells can be followed. Their staining should be bright and homogeneous without affecting cell function. For purposes of monitoring cell proliferation, each time a cell divides the intensity of cell tracking dye should diminish equally between daughter cells. These dyes can be grouped into two different classes. Protein reactive dyes label cells by reacting covalently but non-selectively with intracellular proteins. Carboxyfluorescein diacetate succinimidyl ester (CFSE) is the prototypic general protein label. Membrane intercalating dyes label cells by partitioning non-selectively and non-covalently within the plasma membrane. The PKH membrane dyes are examples of lipophilic compounds whose chemistry allows for their retention within biological membranes without affecting cellular growth, viability, or proliferation when used properly. Here we provide considerations based for labeling cell lines and peripheral blood mononuclear cells using both classes of dyes. Examples from optimization experiments are presented along with critical aspects of the staining procedures to help mitigate common risks. Of note, we present data where a logarithmically growing cell line is labeled with both a protein dye and a membrane tracking dye to compare dye loss rates over 6days. We found that dual stained cells paralleled dye loss of the corresponding single stained cells. The decrease in fluorescence intensity by protein reactive dyes, however, was more rapid than that with the membrane reactive dyes, indicating the presence of additional division-independent dye loss.
摘要：
广义地说,细胞示踪染料是荧光化合物，可与细胞上或细胞内的成分稳定结合，因此可以跟踪标记细胞的命运。它们的染色应该是明亮和均匀的，而不影响细胞功能。为了监测细胞增殖，每次细胞分裂时，子细胞之间的细胞示踪染料的强度应相等地减少。这些染料可以分为两个不同的类别。蛋白质反应性染料通过共价但非选择性地与细胞内蛋白质反应来标记细胞。羧基荧光素二乙酸酯琥珀酰亚胺酯（CFSE）是典型的通用蛋白质标记。膜嵌入染料通过在质膜内非选择性和非共价分配来标记细胞。PKH膜染料是亲脂性化合物的例子，其化学性质允许它们保留在生物膜内而不影响细胞生长。生存能力，或增殖时使用得当。在这里，我们提供了使用两类染料标记细胞系和外周血单核细胞的考虑因素。提供了来自优化实验的示例以及染色程序的关键方面，以帮助减轻常见风险。值得注意的是，我们提供的数据是用蛋白质染料和膜跟踪染料标记的对数生长细胞系，以比较6天的染料损失率。我们发现双重染色细胞与相应的单个染色细胞的染料损失平行。蛋白质活性染料的荧光强度降低，然而,比膜活性染料更快，表明存在额外的非分裂染料损失。
