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Quantitative measurements of ceramides and glycosphingolipids in Th17 and iTreg cells (part-II)

ABSTRACT: Part 2/5: It includes quantitative targeted measurements of sphingolipids (ceramides and glycosphingolipids) in Th17, iTreg, and their paired controls (Th0 cells).

ORGANISM(S): Human Homo Sapiens

TISSUE(S): T-cells

SUBMITTER: Partho Sen

PROVIDER: ST001675 | MetabolomicsWorkbench | Sun Jan 31 00:00:00 GMT 2021

REPOSITORIES: MetabolomicsWorkbench

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Project description:We introduce a microfluidic platform that enables single-cell mass and growth rate measurements upstream of single-cell RNA-sequencing (scRNA-seq) to generate paired single-cell biophysical and transcriptional data sets. Biophysical measurements are collected with a serial suspended microchannel resonator platform (sSMR) that utilizes automated fluidic state switching to load individual cells at fixed intervals, achieving a throughput of 120 cells per hour. Each single-cell is subsequently captured downstream for linked molecular analysis using an automated collection system. From linked measurements of a murine leukemia (L1210) and pro-B cell line (FL5.12), we identify gene expression signatures that correlate significantly with cell mass and growth rate. In particular, we find that both cell lines display a cell-cycle signature that correlates with cell mass, with early and late cell-cycle signatures significantly enriched amongst genes with negative and positive correlations with mass, respectively. FL5.12 cells also show a significant correlation between single-cell growth efficiency and a G1-S transition signature, providing additional transcriptional evidence for a phenomenon previously observed through biophysical measurements alone. Importantly, the throughput and speed of our platform allows for the characterization of phenotypes in dynamic cellular systems. As a proof-of principle, we apply our system to characterize activated murine CD8+ T cells and uncover two unique features of CD8+ T cells as they become proliferative in response to activation: i) the level of coordination between cell cycle gene expression and cell mass increases, and ii) translation-related gene expression increases and shows a correlation with single-cell growth efficiency. Overall, our approach provides a new means of characterizing the transcriptional mechanisms of normal and dysfunctional cellular mass and growth rate regulation across a range of biological contexts.

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Quantitative measurements of ceramides and glycosphingolipids in Th17 and iTreg cells (part-II)

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