Detection of\n>400 Cluster of Differentiation Biomarkers\nand Pathway Proteins in Single Immune Cells by Cyclic Multiplex <i>In Situ</i> Tagging for Single-Cell Proteomic Studies

The identification and characterization of immune cell\nsubpopulations\nare critical to reveal cell development throughout life and immune\nresponses to environmental factors. Next-generation sequencing technologies\nhave dramatically advanced single-cell genomics and transcriptomics\nfor immune cell classification. However, gene expression is often\nnot correlated with protein expression, and immunotyping is mostly\naccepted in protein format. Current single-cell proteomic technologies\nare either limited in multiplex capacity or not sensitive enough to\ndetect the critical functional proteins. Herein, we present a single-cell\ncyclic multiplex <i>in situ</i> tagging (CycMIST) technology\nto simultaneously measure >400 proteins, a scale of >10 times\nthan\nsimilar technologies. Such an ultrahigh multiplexity is achieved by\nreiterative staining of the single cells coupled with a MIST array\nfor detection. This technology has been thoroughly validated through\ncomparison with flow cytometry and fluorescence immunostaining techniques.\nBoth peripheral blood mononuclear cells (PBMCs) and T cells are analyzed\nby the CycMIST technology, and almost the entire spectrum of cluster\nof differentiation (CD) surface markers has been measured. The landscape\nof fluctuation of CD protein expression in single cells has been uncovered\nby our technology. Further study found T cell activation signatures\nand protein–protein networks. This study represents the highest\nmultiplexity of single immune cell marker measurement targeting functional\nproteins. With additional information from intracellular proteins\nof the same single cells, our technology can potentially facilitate\nmechanistic studies of immune responses under various disease conditions.