Propidium iodide

R2J-2D cells were set on a Lab-Tek-Chamber Slide system (Nalge Nunc International, Rochester, NY, USA) and were incubated with 5 µM of Fluorescein Diacetate (FDA, Thermofisher Scientific, Courtaboeuf, France), 1µM Hoechst 33342 (Interchim, Montluçon, France), and 10mg/mL Propidium Iodide (PI) (Interchim) in a 5% CO₂ incubator, 37 °C, 20 min. After staining, cells were installed on the microscope incubation chamber with a controlled atmosphere at 37 °C and 5% CO₂ (POC Chamber, Pecom, Erbach, Germany). Images were collected with a Leica TCS SP2 AOBS (Acoustico Optical Beam Splitter) inverted laser scanning confocal microscope equipped with an ×63 oil immersion objective (HCX PL APO 63.0× 1.40). Laser excitation and emission (adjusted with AOBS) were, respectively, 351–364/425–485 nm for Hoechst, 488/500–540 nm for FDA, and 543/600–650 nm for PI. Confocal pinhole (Airy units) was 1 for all channels. Each experiment was performed on a randomly chosen field. Raw image merging was obtained by LCS Lite software (Leica LCS, version 2.61, Overlay).

To discriminate viability of islet cells from MSCs, MSCs were stained with PKH67 before seeding islets, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA; λexc\λem = 490/504 nm). After cytokine exposure, islets co-cultured with MSCs were washed with PBS, dissociated with trypsin 1× and collected in PBS for a cytometric analysis of single-cell suspension. The two cell types were stained with 1.5 × 10^− 10 mol mL^− 1 of propidium iodide (PI) (Interchim, Montluçon, France), (λex/λem = 545/630) allowing identification of the necrotic cells. Cells labelled with PI and PKH67 were considered as necrotic MSCs, unlabelled cells as viable islet cells and cells stained with PI alone as necrotic islet cells. The measurements were carried out using a FACS Calibur (BD Bioscience, San Jose, CA, USA) instrument.