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Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages

Citation

Ulbricht, Carolin et al. (2021), Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages, Dryad, Dataset, https://doi.org/10.5061/dryad.cc2fqz63d

Abstract

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the FRET-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial and detrimental (auto)immunity.

Methods

Absolute calcium values calculated per cell out of microscopy data. The data were collected as fluorescent lifetime values with a time correlated single photon counting device in each pixel of an image. The images have been processed with Imaris software (bitplane) for segmentation of single cells/objects. Values were exported over time per object in excel spreadsheets. Out of each fluorescent lifetime value, absolute calcium concentration was calculated according to the formula given in the original publication.

Usage Notes

A README file is available, explaining variables.

Funding

Deutsche Forschungsgemeinschaft, Award: TRR130