IL-1 [38]) and tissue integrity (e.g. Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is usually co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity. Conclusions/interpretation Our results indicate Rabbit Polyclonal to Akt (phospho-Tyr326) that islet macrophages detect ATP as a proxy transmission for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size. recombinase in myeloid cell-specific promoters: ((B6, stock no. 004781; JAX laboratories); Tg(recombinase in Imidazoleacetic acid myeloid cell-specific promoters with mice expressing the floxed Ca2+ indication GCaMP3 (observe above). Mice ( 3 months aged, both sexes) were euthanised, and pancreatic tissue slices were processed as explained above. Living tissue slices made up of GCaMP3-labelled macrophages were placed in a perfusion chamber and immersed in HEPES-buffered answer. Glucose was added to the buffered answer to give a basal glucose concentration of 3 mmol/l, unless otherwise specified. All stimuli Imidazoleacetic acid were bath applied. Throughout the study we used the non-hydrolysable ATP agonist ATPS (adenosine 5-(3-thiotriphosphate; Tocris Biosciences, Bristol, UK). Antagonists were left to equilibrate with receptors for 5 min before activation with an agonist. For [Ca2+]i imaging, a Z-stack of ~15C30 confocal images was acquired every 8 s using a Leica SP5 confocal laser-scanning microscope. [Ca2+]i responses in pancreatic macrophages were quantified as the AUC of individual traces of GCaMP3 fluorescence intensity (expressed as switch in fluorescence intensity compared with baseline fluorescence Imidazoleacetic acid Imidazoleacetic acid [F/F]) during the application of stimuli. To be included in the analyses, [Ca2+]i responses had to be reproducible in three or more pancreatic slices. We further analysed and quantified pseudopodia movement and velocity using the ImageJ plugin MTrackJ Imidazoleacetic acid (https://imagescience.org/meijering/software/mtrackj/, accessed 14 October 2016.). For immunohistochemical staining after [Ca2+]i imaging, slices were processed as explained in ESM Methods (Immunohistochemistry section). Confocal imaging Confocal images (pinhole = 1 airy unit) of randomly selected islets were acquired on a Leica SP5 confocal laser-scanning microscope with 40 magnification (NA = 0.8). Macrophages were reconstructed in Z-stacks of 15C30 confocal images (step size = 2.5C4.0 m) and analysed using ImageJ (version 1.51h; http://imagej.nih.gov/ij). Using confocal images, we established the location of macrophages within islets (endocrine) or in acinar regions (exocrine). Experiments were not blinded. To prevent bias, we used an automated method in ImageJ to segment different pancreas regions based on DAPI staining to determine macrophage positions. Circulation cytometry Islet macrophages were sorted based on the viable, GFP+ and F4/80+ labelled cells. For non-macrophage internal controls, islet cells were also sorted based on the viable GFP?, F4/80? population. Observe ESM Methods for additional details. RT-PCR RNA was extracted from FACS sorted islet macrophages and non-macrophage internal controls using RNeasy mini kit (Qiagen, Valencia, CA), and cDNA was prepared using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). cDNA products were pre-amplified 10 cycles using the TaqMan pre-amp grasp mix (Applied Biosystems). PCR reactions were run using TaqMan gene expression assays (Applied Biosystems) in a StepOnePlus Real-Time PCR System (Applied Biosystems). Relative quantification of gene expression was done based on the equation relative quantification = 2?Ct 1,000,000 where Ct is the.