PT  - JOURNAL ARTICLE
AU  - Carolin Ulbricht
AU  - Ruth Leben
AU  - Asylkhan Rakhymzhan
AU  - Frank Kirchhoff
AU  - Lars Nitschke
AU  - Helena Radbruch
AU  - Raluca A Niesner
AU  - Anja E Hauser
TI  - Intravital quantification of absolute cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stages
AID  - 10.1101/2019.12.13.872820
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2019.12.13.872820
4099  - http://biorxiv.org/content/early/2020/02/13/2019.12.13.872820.short
4100  - http://biorxiv.org/content/early/2020/02/13/2019.12.13.872820.full
AB  - Development, function and maintenance of lymphocytes largely depend upon the cellular mobilization and storage of Ca2+ ions. In B lymphocytes, the absolute amount of calcium mobilized and retained after cell signaling remains unknown, athough it is a crucial part of their selection within germinal centers and differentiation into plasma cells. Here, we introduce the novel reporter mouse strain YellowCaB that expresses the genetically encoded calcium indicator TN-XXL in CD19+ lymphocytes. The construct consists of the electrondonor fluorophore eCFP and the acceptor citrine, linked by a calcium sensitive domain. Its conformation and therefore donor quenching is directly linked to cytosolic calcium concentrations. By combining intravital two-photon fluorescence lifetime microscopy with our numerical approach for phasor-based analysis, we are able to extract absolute cytoplasmic calcium concentrations in activated B cells for the first time in vivo. We show that calcium concentrations in B cells are highly dynamic and fluctuations persist in extrafollicular B cells with functional relevance.