title: An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells.
abstract: Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to combine mitochondrial Ca(2+) measurements with nuclear translocation. In conclusion, this method enables the high throughput study of spatiotemporal patterns of Ca2(+) signals in T cells responding to different stimuli.
authors.affiliation: FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK.
authors.forename: Joana
authors.initials: J
authors.lastname: Cerveira
authors.affiliation: FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK.
authors.forename: Julfa
authors.initials: J
authors.lastname: Begum
authors.affiliation: Immuno Surveillance Laboratory London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn, London WC2A 3LY, UK.
authors.forename: Rafael
authors.initials: R
authors.lastname: Di Marco Barros
authors.affiliation: Immunobiology Laboratory London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn, London WC2A 3LY, UK.
authors.forename: Annemarthe G
authors.initials: AG
authors.lastname: van der Veen
authors.affiliation: Flow Cytometry Core Facility, Newcastle Biomedicine, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK; FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK. Electronic address: andrew.filby@newcastle.ac.uk.
authors.forename: Andrew
authors.initials: A
authors.lastname: Filby
mesh_terms: D017252:CD3 Complex / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology*; D008853:Microscopy / Q000379:methods; D008928:Mitochondria / Q000378:metabolism; D062206:Spatial Analysis; D062211:Spatio-Temporal Analysis; D013601:T-Lymphocytes / Q000378:metabolism* / Q000502:physiology*
chemical_list: D017252:CD3 Complex; D002118:Calcium
keywords: Calcium flux; Imaging flow cytometry; NFAT translocation; T cells