Fig. 1.
[Ca2+]i imaging of pancreas macrophages in situ. (a) Cre-lox model used to generate the [Ca2+]i indicator GCaMP3 under the control of myeloid-specific promoters. (b, c) Living pancreatic tissue cut using a vibroslicer (b) and placed in a recording chamber (scale bar, 100 μm) (c). Arrows denote location of a pancreatic islet. (d) Confocal image of a fixed pancreatic slice from a Csf1r-Cre–GCaMP3 mouse showing GCaMP3 (green) and IBA1 (red) immunostaining, and DAPI (blue) staining of nuclei. Scale bar, 50 μm. Zoomed images of islet macrophages are also shown (scale bar, 10 μm). (e) Quantification of the proportion of IBA1-positive cells within the population of GCaMP3+ cells in slices (> 75 macrophages pooled from four sections in three mice per group). Box and whisker plot shows the median (horizontal line) with the lower and upper quartiles depicted by the bottom and top edge of the box, respectively. (f) Confocal image taken from a live video recording during [Ca2+]i imaging of macrophages in a living slice from a GCaMP3+ mouse. A maximal projection of a Z-stack of ~40 images is shown. The image shows islet backscatter (grey) and GCaMP3-labelled macrophages (green), with macrophages labelled 1–4. (g) Three sequential higher magnification images of macrophage ‘number 1’, shown in the dotted box in (f), showing the [Ca2+]i response to stimulation with ATP (100 μmol/l). The pseudocolour scale indicates the level of fluorescence intensity. Time points are before, during and after stimulation with ATP. Scale bar, 10 μm. (h) Traces of [Ca2+]i responses in macrophages 1–4 shown in (f). Arrows indicate time points before, during and after stimulation with ATP at which the images in (g) were taken. AU, arbitrary units