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A robust protocol for detecting highly expressed and rare intracellular cytokine protein expression in CD4 T Cells and monocytes using TotalSeq cell surface and intracellular antibodies 2447

Year: 2025
Type: article
Abstract: Description Cytokines are small, secreted proteins that are essential for immune-cell regulation during a variety of immune responses including infection, auto-immunity and therapeutics. Characterizing immune cell types responsible for cytokine production is important to elucidate the mechanism of a given immune response and is frequently done by intracellular flow cytometry. Single-cell NGS provides similar single-cell resolution to flow cytometry and enables the ability to profile the transcriptome and a greater number of cell surface and intracellular proteins simultaneously. In this study we profiled fresh PBMCs using the 10X Genomics Chromium Fixed RNA Profiling kit with a TotalSeq Universal cocktail and 28 intracellular antibodies to cytokines, chemokines and interferons. CD4+ T cells were magnetically enriched and stimulated for 6 hours using PMA, Ionomycin and Brefeldin A. CD14+ monocytes were magnetically enriched and stimulated overnight with either GM-CSF or IFN-gamma, followed by LPS, R848 and Brefeldin A. Cells were processed by the TotalSeq Surface and Intracellular Cell Staining protocol. RNA, cell surface and intracellular proteins were characterized for each sample treatment. Our data demonstrate that gene expression was not significantly altered with the intracellular staining protocol. Both highly expressed (e.g. IL-2, TNF-alpha, IFN-gamma) and rare (e.g. IL-4, IL-22, IL-12p40) cytokines were detected in each cell type demonstrating assay specificity and sensitivity. Funding Sources Funded by BioLegend Topic Categories Technological Innovations in Immunology (TECH)
DOI: 10.1093/jimmun/vkaf283.364
Language: English
Open Access status: bronze
OpenAlex ID: W4416448807
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