BACKGROUND Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study.

中文翻译：

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在兽医相关物种的细胞分析中克服流式细胞术局限性的方法。

背景技术流式细胞术是用于在单细胞水平上对白细胞亚群进行多参数分析的强大工具。最近的进展极大地增加了流式细胞术中荧光染料标记的抗体的数量。特别是，具有不同激发和发射光谱的可用荧光染料的增加，再加上带有多个激光器的新型多色流式细胞仪，已经增强了白细胞和其他细胞类型的多维表达数据的生成。但是，由于缺乏大多数动物物种的试剂，这些进展主要有益于人类或小鼠细胞样品的分析。对重要兽医，农业，野生动植物和其他动物物种的流式细胞仪分析仍然受到一些技术限制的阻碍，即使除了老鼠以外的其他动物物种也可以充当特定人类生理和疾病的更准确模型。结果在这里，我们介绍了经过时间检验的方法，这些方法是我们实验室在绵羊白细胞的多参数流式细胞术分析中经常使用的方法。讨论的方法将适用于大多数动物物种的细胞分析，包括通过共价结合或Fc定向标记（Zenon™技术）对抗体的直接修饰，标记的第二抗体和其他第二步试剂，标记的受体配体和抗体与物种交叉反应。结论使用完善的技术方法，可以大大增加每个细胞样品通过流式细胞术分析的参数数量，能够对稀有样品进行多维分析，并对兽医和其他不常分析的物种提供重要的洞察力。通过最大化每个细胞样本中的信息，多色流式细胞术可以减少研究中所需的动物数量。