Imagem camera

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 ESM Methods.

Bacterial OMVs were stained with the green, fluorescent membrane dye PKH67 (Sigma, ON, Canada, cat. no. MINI67) at 1 μM final concentration in Diluent C (Sigma) for 5 min. The stained OMVs were then pelleted at 30,000g for 1 h in a TL-100 Optima Ultracentrifuge (Beckman) using rotor TLA 100.4. PKH67-labeled OMVs were washed in HBSS 1X (Wisent, QC, Canada, cat. no. 311-513-CL) to get rid of the free dye and then incubated with the fluorescently labeled HCT116 cells. HCT116 cells were labeled with the CellTracker Red CMTPX Dye (Invitrogen, ON, Canada, cat. no. C34552) for 30 min at 37°C in darkness and washed with PBS.
After incubation for the indicated times, trypsin was used for cell dissociation followed by centrifugation at room temperature for 5 min. Cells were then resuspended in 100 μl 2% PFA and kept for 15 min at room temperature to allow fixation. Cell deposition on slides was performed using cytospin. One drop of Gold Antifade Mountant with DAPI (SlowFade, ON, Canada, cat. no. S36942) was applied directly to the slide, which was then sealed with varnish and imaged using a Wave FX-Borealis—Leica DMI 6000B (Quorum Technologies) microscope (63X) with ImagEM camera (Hamamatsu, 512x512 pixels) at the CHUL–Université Laval Bio-imaging platform. The images were processed with the Volocity 4.2.1 software (Quorum Technologies).

Isolated islets were loaded with 5 μm Fura-2AM (Dojindo, Japan) at 37 °C for 30 min, placed in a heat-controlled chamber on the stage of an inverted microscope kept at 37 °C, superfused with KRBB containing 2.8 mM glucose, 0.2% BSA, and 10 mM Hepes (pH 7.4), and subsequently exposed to the buffer containing 16.7 mM glucose and then to buffer containing 2.8 mM glucose with 30 mM KCl. The islets were excited successively at 340 and 380 nm, and the fluorescence emitted at 510 nm was captured by an Olympus IX-70 microscope coupled to an ImagEM camera (Hamamatsu Photonics, Hamamatsu, Japan). The images were analyzed with the AQUACOSMOS analyzing system (Hamamatsu Photonics). The 340 nm (F340) and 380 nm (F380) fluorescence signals were detected every 20 seconds, and ratios (F340/F380) were calculated.