Kasprowicz, Richard Mark Robert, Rand, Emma, O'Toole, Peter John orcid.org/0000-0001-5295-2001 et al. (1 more author) (2018) A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses. Scientific Reports. ISSN 2045-2322
Abstract
Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2018 |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Hull York Medical School (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 16 May 2018 14:30 |
Last Modified: | 26 Nov 2024 00:38 |
Published Version: | https://doi.org/10.1038/s41598-018-26172-3 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1038/s41598-018-26172-3 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:130857 |
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