A Mine Is a Terrible Thing to Waste: High Content, Single Cell Technologies for Comprehensive Immune Analysis

Abstract

In recent years, an incredible variety of single cell technologies have become available to analyze immune responses. These technologies include polychromatic flow cytometry, mass cytometry, highly multiplexed single cell qPCR, RNA sequencing, microtools, and high‐resolution imaging. In this article, we review these platforms, describing their power and limitations for comprehensive analysis of the immune system. We relate the properties of these technologies to the various cellular states relevant to an immune response, in order to address which technologies are most appropriate for which settings.

Human immune system variation

“…This technique enables single cell analysis by using fluorescently labelled antibodies measuring up to 30 simultaneous parameters in millions of individual cells in the most technically specialized laboratories 98 ; however, most flow cytometric protocols detect 15 or fewer simultaneous parameters. The speed and versatility of the technology and the ability to sort viable cells makes flow cytometry a cornerstone technology in immunology research.…”

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images

“…Despite its feasibility, reproducibility and affordability in multiparametric analyses, one of the major drawbacks of flow cytometry is exactly its basic principle: the fluidic stream. 9 The crucial need for sample disaggregation leads to sample loss at post-analysis and the abrogation of the natural architecture of samples, which is critical when analyzing rare (on occasion, small) biological samples and for morphological-based analyses of adherent cell cultures and/or whole tissues. 9 Therefore, imaging analyses software constitute a fast, accurate and cost-effective solution, as they allow the preservation of the natural architecture of cells.…”

“…9 The crucial need for sample disaggregation leads to sample loss at post-analysis and the abrogation of the natural architecture of samples, which is critical when analyzing rare (on occasion, small) biological samples and for morphological-based analyses of adherent cell cultures and/or whole tissues. 9 Therefore, imaging analyses software constitute a fast, accurate and cost-effective solution, as they allow the preservation of the natural architecture of cells. In fact, the application of digital technology to slide-format (for fixed samples) or plate-format (for live-cell imaging) enables quantitative determination of DNA content, as well as multiparametric analyses encompassing morphological, cellular and sub-cellular features of single cells.…”

Untitled

“…This method allows analyzing single cells using fluorescently labeled antibodies and cell compartment markers by measuring up to 30 parameters simultaneously in hundreds of thousands and millions of cells [139]. This analysis yields a wealth of reliable information about differentiation and properties of cell subpopulations.…”

“…Information obtained with the abovementioned techniques can be of great importance for fundamental immunology. If discovered, novel immunity markers will be adapted for use in simple diagnostic procedures, including multiplex immunoassays [140], IGRA-tests, polymerase chain reaction assays, flow cytometry [139] or mass spectrometry [141].…”