Figure 3. Representative Ca²⁺ signaling images in Peyer’s patches of a YC3.60^flox/CD19-Cre mouse.

(a) Image of Peyer’s patches of a YC3.60^flox/CD19-Cre mouse after staining for CD4 and PD-1. YC3.60⁺ cells (green), CD4⁺ T cells (Blue), and PD-1⁺ cells (red) are shown (left). PE-conjugated anti-CD4 mAbs and Alexa647-conjugated anti-PD-1 mAs were intravenously injected 30 min before observation. Representative Ca²⁺ signaling image in Peyer’s patches of a YC3.60^flox/CD19-Cre mouse (right). Only ratiometric images (YFP/CFP at excitation of 458 nm) are shown. Cells exhibiting FRET signals are indicated by arrows. Results are representative of at least three independent experiments (n = 3 mice). Scale bars, 25 μm. (b) Time courses of intracellular Ca²⁺ fluxes. Ratiometric intensities (YFP/CFP at excitation of 458 nm) of indicated cells in (a) were measured for 10 min with a no-delay program. Frame = 1129. (c) Z-stack analysis of intracellular Ca²⁺ concentrations of B cells in Peyer’s patches. Intravital imaging of Peyer’s patches was performed using two-photon microscopy. Ratiometric images (YFP/CFP at excitation of 840 nm) are shown. Z-stack images of 2-μm intervals up to a depth of 200 μm were obtained. Only representative images are shown. The follicle is indicated by a broken line. IF: interfollicular region. (d) Three-dimensional (3D) structure of B cells with intracellular Ca²⁺ concentrations in Peyer’s patches. 3D images based on Z-stack images (c) were obtained using Nis Elements software. (e) Distribution of intracellular Ca²⁺ concentrations of B cells of the indicated Z-stack images. n = 50.