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

Figure 2. B cell receptor (BCR) stimulation specifically leads to calcium mobilization in YellowCaB cells in vitro.

(a) Confocal measurement of Förster resonance energy transfer (FRET) duration (ΔR/R > 0) in non-perfused primary polyclonal YellowCaB cells after addition of 10 µg/ml anti-IgM-F(ab)2 (black) and ionomycin control (gray). Data representative for at least 35 single cells in four independent experiments. (b) Confocal measurement of FRET signal change after repeated addition of anti-IgM-F(ab)2 to perfused primary polyclonal YellowCaB cells. Data representative for at least 50 cells out of five independent experiments. (c) Confocal measurement of FRET signal change after addition of anti-IgM-F(ab)2 to perfused primary polyclonal YellowCaB cells following stimulation with anti-CD40 antibody and ionomycin as positive control. Examples of transient cytoplasmic (blue), intermediate (gray), and sustained calcium mobilization shown, area under the curve compared. Data representative for 26 cells out of two independent experiments. (d) Resulting FRET curve out for n = 7 primary polyclonal YellowCaB cells perfused with toll-like receptor (TLR)9 stimulator cytosine phosphate guanine (CpG) in Ringer solution and subsequent addition of anti-IgM-F(ab)2. (e) Mean FRET signal change over time after addition of TLR4 or TLR9 stimulation in combination with BCR crosslinking by anti-IgM-F(ab)2 in perfused polyclonal YellowCaB cells. n = 12 (top) and n = 8 (bottom), one-way ANOVA. Error bars: SD/mean.

Figure 2.

Figure 2—figure supplement 1. Confocal measurement and plot of TN-XXL ΔR/R over time.

Figure 2—figure supplement 1.

(a) In an open perfused system (Krebs–Ringer solution 6 mM Ca2+), single cells were stimulated with indicated concentrations of B cell receptor anti-light- or anti-heavy chain antibody (arrows). Antibodies were added directly to the imaging chamber volume. Perfusion pump was switched off during stimulation and switched on again at indicated time points (dashed lines). (b) Open perfusion of single cells with 6 mM Ca2+ Krebs–Ringer solution or 6 mM Ca2+ Krebs–Ringer plus reagents indicated. Here, the stimulation was performed under continuous flow via changing the reservoir of the afferent buffer solution. (c) Ratiometric measurement over time of isolated YellowCaB cells after stimulation with CXCL12. Experimental set-up as described in (b). (d) Absolute calcium concentration measured using fluorescence lifetime imaging after CXCL12 stimulation of ex vivo lipopolysaccharide-induced plasmablasts at indicated time points.