Pancreatic islets from reporter mice expressing Nnat-eGFP were isolated as above, with Ca2+ imaging of whole islets performed after loading the cytosol with 2 μmol/l Cal-590 AM (Stratech, UK). Images were captured on an Axiovert microscope (Zeiss, Germany) equipped with a ×10, 0.3–0.5 numerical aperture (NA) objective and a ImagEM camera (Hamamatsu, Japan) coupled to a Nipkow spinning-disk head (CSU-10, Yokogawa, Japan) and illuminated at 490 nm or 530 nm.
For experiments involving global Nnat null mice [35 (link)], islets were incubated with 4.5 μmol/l Cal-520 AM (Stratech), and imaging was performed on a Nikon (Japan) Eclipse Ti microscope equipped with a ×40/1.2 NA oil objective and an ibidi heating system. Cal-520 AM was excited with a 491 nm laser line and emitted light filtered at 525/50 nm. Images were acquired with an ORCA-Flash 4.0 camera (Hamamatsu) and Metamorph software (Molecular Devices). Pearson-based connectivity and correlation analyses in an imaged islet were performed with Ca2+ signals smoothed, binarised and analysed as described in the ESMMethods .
For experiments involving global Nnat null mice [35 (link)], islets were incubated with 4.5 μmol/l Cal-520 AM (Stratech), and imaging was performed on a Nikon (Japan) Eclipse Ti microscope equipped with a ×40/1.2 NA oil objective and an ibidi heating system. Cal-520 AM was excited with a 491 nm laser line and emitted light filtered at 525/50 nm. Images were acquired with an ORCA-Flash 4.0 camera (Hamamatsu) and Metamorph software (Molecular Devices). Pearson-based connectivity and correlation analyses in an imaged islet were performed with Ca2+ signals smoothed, binarised and analysed as described in the ESM