An imaging flow cytometry-based approach to measuring the spatiotemporal calcium mobilisation in activated T cells

• Imaging Flow Cytometry can be used to measure the spatiotemporal Ca2 + flux in activated T cells.
• We can simultaneously measure fluorescently tagged transcription factors such as NFAT.
• We have uncovered a novel dynamic interplay between the ER and mitochondrial Ca2+ stores in activated T Cells.

Calcium ions (Ca2 +) 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 Ca2 + from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca2 + 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 Ca2 + 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 Ca2 + 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 Ca2 + indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca2 + 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 Ca2 + 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 Ca2 + after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca2 +. 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 Ca2 +. Moreover, by generating an NFAT–GFP expressing Jurkat line, we were able to combine mitochondrial Ca2 + 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.