Figure 5. Representative Ca²⁺ signaling images in the spleen of an OVA/alum-immunized YC3.60^flox/CD19-Cre mouse.

(a) Images of Ca²⁺ signaling in the spleen of a non-immunized mouse and primary-immunized YC3.60^flox/CD19-Cre mouse. YC3.60^flox/CD19-Cre mice were immunized with OVA/alum; after 14 days, analysis of spleens of the mice by intravital imaging was performed using confocal microscopy. Images were obtained every 2 s. Representative Ca²⁺ images based on the ratio (YFP/CFP at excitation 458 nm) are shown (n = 3). (b) Ratiometric intensities (YFP/CFP at excitation 458 nm) of splenic B cells of control (alum only) and OVA-immunized mice. P < 0.01 (t-test). Bars denote mean values. n = 52. (c) Distribution of time-integrated intracellular Ca²⁺ concentrations of B cells of non-immunized and OVA-immunized mice. n = 30, frame = 13 (alum) and n = 16, frame = 90 (OVA/alum). Percentages of cells: ratios > 1.2 are indicated. Representative data of three mice are shown. (d) Images of Ca²⁺ signaling in the spleen of a secondary-immunized YC3.60^flox/CD19-Cre mouse. YC3.60^flox/CD19-Cre mice were immunized with OVA/alum and boosted on day 30; after 7 days, analysis of the spleens of the mice was conducted with intravital imaging. Representative Ca²⁺ images based on the ratio (YFP/CFP at excitation of 458 nm) at indicated time points are shown. Cells exhibiting FRET signals are indicated by arrows. Results are representative of at least three independent experiments (n > 3 mice). (e) Time course of intracellular Ca²⁺ fluxes. Ratiometric intensities (YFP/CFP at excitation of 458 nm) of indicated cells in (a) were measured every 5 s for 10 min. (f) Ratiometric intensities (YFP/CFP at excitation of 458 nm) of splenic B cells of non-immunized and OVA-immunized mice. P < 0.01 (t-test). Bars denote mean values. n = 50. (g) Distribution of time-integrated intracellular Ca²⁺ concentrations of B cells of non-immunized and OVA-immunized mice. n = 50, frame = 61. Percentages of cells: ratios > 1.2 are indicated.