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 μM Cal-590 AM (Stratech). Images were captured on an Axiovert microscope (Zeiss, Germany) equipped with a 10x 0.3–0.5 NA objective, a Hamamatsu ImagEM camera coupled to a Nipkow spinning-disk head (CSU-10, Yokogawa UK Ltd) and illuminated at 490 nm or 530 nm.
For experiments involving global Nnat null mice [45 (link)], islets were incubated with 4.5 μM Cal-520 AM (Stratech), and imaging performed on a Nikon Eclipse Ti microscope equipped with a 40x/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 Device). Pearson-based connectivity and correlation analyses in an imaged islet were performed with Ca2+ signals smoothed, binarised and analysed as described inESM methods .
For experiments involving global Nnat null mice [45 (link)], islets were incubated with 4.5 μM Cal-520 AM (Stratech), and imaging performed on a Nikon Eclipse Ti microscope equipped with a 40x/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 Device). Pearson-based connectivity and correlation analyses in an imaged islet were performed with Ca2+ signals smoothed, binarised and analysed as described in
