Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

成像是研究蛋白质表达的有力方法，在提供原位空间信息方面比其他方法更具优势在单个单元格级别。使用免疫荧光和共聚焦显微镜，可以获得蛋白质亚细胞分布的详细信息。尽管来自不同组织起源的贴壁细胞相对容易准备用于成像应用，但是来自造血细胞的非贴壁细胞由于其与表面的附着力差以及随后损失了大量细胞而提出了挑战。这些细胞类型仍然代表着人类蛋白质组的重要组成部分，并表达在贴壁细胞类型中未表达的基因。在细胞作图的时代，克服悬浮细胞在成像应用中的挑战将使造血细胞的系统分析成为可能。在这项工作中，我们成功建立了用于制备悬浮细胞系的免疫荧光方案，粘附表面上的外周血单核细胞（PBMC）和人血小板。该协议基于具有自动样品制备功能的多孔板格式，可用于强大的高通量成像应用。与共聚焦显微镜相结合，该协议可以系统地探索蛋白质在所有主要亚细胞结构中的定位。