Binan Loïc, Roy Joannie, Costantino Santiago
Research center, Maisonneuve-Rosemont Hospital, Montreal, Canada.
Department of ophthalmology, University of Montreal, Montreal, Canada.
Bio Protoc. 2019 Nov 20;9(22):e3428. doi: 10.21769/BioProtoc.3428.
Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or intracellular protein localization. We previously introduced a method termed Cell Labeling via Photobleaching (CLaP) for the efficient tagging of cells chosen for visual criteria. Here we describe a new protocol for capturing such cells using ferromagnetic beads termed single-cell magneto-optical capture (scMOCa). This technology is especially useful when the number of target cells represents an extremely low fraction of the total population (potentially one single cell), a situation in which conventional sorting techniques like fluorescent or magnetic activated cell sorting (F/MACS) cannot provide satisfactory results in terms of capture efficiency and specificity. scMOCa uses the lasers of a confocal microscope to photobleach and crosslink biotin-4-fluorecein molecules to cell membranes. Streptavidin coated magnetic beads then adhere to biotin moieties and a magnet allows the capture of illuminated cells. By precisely controlling liquid volumes and spacing between the different parts of a simple setup, high cell selectivity and capture efficacy can be achieved. scMOCA allows visual selection and isolation of any number of cells in a microscopy field and captured cells remain viable to generate new colonies of chosen phenotypes for downstream analyses.
仅基于可观察特征从大型异质群体中捕获单个细胞，对于许多细胞生物学应用来说是必要的，但仍然是一项重大技术挑战。我们提出的方案允许分离出因其形状、迁移速度、与其他细胞的接触或细胞内蛋白质定位而被选择的有活力且代谢活跃的细胞。我们之前引入了一种称为光漂白细胞标记（CLaP）的方法，用于对根据视觉标准选择的细胞进行有效标记。在这里，我们描述了一种使用铁磁珠捕获此类细胞的新方案，称为单细胞磁光捕获（scMOCa）。当目标细胞数量在总群体中所占比例极低（可能只有一个单细胞）时，这项技术特别有用，在这种情况下，传统的分选技术，如荧光或磁性激活细胞分选（F/MACS），在捕获效率和特异性方面无法提供令人满意的结果。scMOCa使用共聚焦显微镜的激光对细胞膜上的生物素 - 4 - 荧光素分子进行光漂白和交联。然后，包被链霉亲和素的磁珠会附着在生物素部分上，并且一个磁铁可以捕获被照亮的细胞。通过精确控制一个简单装置不同部分之间的液体体积和间距，可以实现高细胞选择性和捕获效率。scMOCA允许在显微镜视野中对任意数量的细胞进行视觉选择和分离，并且捕获的细胞保持活力，以产生用于下游分析的所选表型的新菌落。
