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. 2021 Mar 22;10:e56020. doi: 10.7554/eLife.56020

Figure 6. Antigen dependency of calcium elevation in germinal center (GC) and extrafollicular B cells.

(a) Top panel: absolute calcium concentrations measured in antigen (AG)-specific GC B cells before and after in vivo injection of NP-BSA. Exemplary results (left) and pooled data from three imaged mice (right). Bottom panel: absolute calcium concentrations measured in AG-specific GC B cells before and after in vivo injection of the Bruton’s tyrosine kinaseinhibitor ibrutinib, followed by injection of NP-BSA. Exemplary results (left) and pooled data from three imaged mice (right). (b) z-stack of intravitally imaged lymph node with GC (white line) and subcapsular sinus (indicated by SHG, blue). CD169+ macrophages (red, contacts [yellow], YellowCaB cells [green]). Size 500 × 500 × 78 µm. Scale bar 60 µm. (c) Top: Fluorescence lifetime imaging measurement of mean absolute calcium concentration of YellowCaB cells showing no (–), transient (+), or strong (++) overlap with CD169+ signal. n = 1000, ANOVA analysis, mean and SD. Bottom: Single-cell track of a YellowCaB cell making transient contact to a macrophage; blue: colocalization intensity (AU); black: change of absolute calcium concentration.

Figure 6.

Figure 6—figure supplement 1. Calcium concentration change detected by in vivo fluorescence lifetime imaging measurements over time, exemplary single germinal center (GC) B cell tracks, before and after injection(s) of compounds.

Figure 6—figure supplement 1.

(a) Absolute calcium measured over time in GC B cell tracks before (left segment, n = 15) and after (right segment, n = 20) intravenous (i.v.) injection of NP-BSA. (b) Absolute calcium measured over time in GC B cell tracks before (left segment, n = 15) and after (middle segment, n = 22) i.v. injection of ibrutinib (gray arrow), followed by injection of NP-BSA (right segment, n = 11, black arrow). Space between dashed lines represents dynamic range of TN-XXL.
Figure 6—figure supplement 2. Absolute calcium concentration of fluorescence lifetime imaging measured after NP-BSA stimulation of ex vivo lipopolysaccharide-induced plasmablasts.

Figure 6—figure supplement 2.

Mann–Whitney test, SEM.
Figure 6—figure supplement 3. Colocalization histogram and exponential fit for analysis of colocalization between CD169+ macrophages and extrafollicular YellowCaB cells.

Figure 6—figure supplement 3.

Colocalization intensity was determined via setting Intensity thresholds for red (macrophages) and green (YellowCaB cells) and calculation of overlap in each pixel. Colocalization intensity of <1 was considered non-colocalized, and colocalization intensity >1 was considered colocalized with weak (+), intermediate (++), or strong (+++) contact depending on decay rates (<25% for ++, <10% for +++ contact) of biexponential fit.