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.
Source data for flow cytometric Analysis, in vitro confocal imaging, ratiometric in vivo Imaging and fluorescence lifetime in vivo Imaging are deposited at Dryad Digital Repository 10.5061/dryad.cc2fqz63d. Analyzed absolute calcium concentration for all cells measured out of 5 experiments have also been deposited there. Source code for phasor based analysis of fluorescence lifetime data has been provided with full submission upload and will be made available to the public via github after publication.
Intravital quantification of cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stagesDryad Digital Repository, doi:10.5061/dryad.cc2fqz63d.
- Helena Radbruch
- Anja E Hauser
- Raluca A Niesner
- Anja E Hauser
- Lars Nitschke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: The study was approved by the Berlin Landesamt für Gesundheit und Soziales under the registration # G00158/16. All surgeries and experimental procedures were conducted following the principle of minimization of suffering and 3R means were used where possible.
- Michael L Dustin, University of Oxford, United Kingdom
- Received: February 14, 2020
- Accepted: March 19, 2021
- Accepted Manuscript published: March 22, 2021 (version 1)
© 2021, Ulbricht et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.