Abstract
In this study we demonstrate the feasibility of direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors following a brief ex vivo stimulation. Cytokine-secreting cells were identified using cell surface “catch” reagents and single cell data were obtained by sorting of individual cytokine-secreting cells into 96 well plates containing lysis buffer followed by analysis using ultrasensitive immunoassays for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). CD8 cells negative for cytokine production, as determined by the cell surface catch reagents were used as negative controls. Furthermore, studies were undertaken to compare the mean fluorescence intensity (MFI) values of cytokine staining by flow cytometry with the quantification of cytokines using the current method. This study demonstrates that it is feasible to quantify cytokines from individual primary cells. A shift from qualitative to quantitative determinations of cytokine protein levels in single cells will permit more precise and reproducible studies of heterogeneity in the immune system and can be accomplished with readily available instrumentation.
Highlights
The increasing awareness of cellular heterogeneity among cell populations that might appear uniform by morphologic or phenotypic characteristics has prompted techniques for analysis of single cells [1, 2]
We report the ultrasensitive detection of pro-inflammatory cytokines IFN-γ and TNF-α in single CD8 T cells in human peripheral blood using only commercially-available instrumentation
Using the power of flow cytometry cell sorting together with an automated Single Molecule Array (SiMoA) platform, we demonstrated protein quantification from single human primary cells that can be performed without involving PCR, custom made microfluidics, or complex modeling methodologies
Summary
The increasing awareness of cellular heterogeneity among cell populations that might appear uniform by morphologic or phenotypic characteristics has prompted techniques for analysis of single cells [1, 2] This heterogeneity has been observed using technologies such as flow and mass cytometry [3, 4], the study of single cell isolates from the heterogenous populations has involved primarily genomic and transcriptomic approaches. Most immunoassays used to study cellular proteins, such as ELISA, Luminex bead arrays, and Western blots, have sensitivities in the picogram to high nanogram per ml range which are too insensitive to detect and quantify proteins from individual cells and require quantification of analytes to be performed on large number of cells which are assumed to be homogeneous These conventional methods are accessible and simple but can incorrectly describe the distribution of behavior among individual cells in the sample.
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