Low melt agar

To evaluate the protein expression, immunocytochemistry (ICC) technique was applied. After 5 days of coculture, the islets in all groups were fixed in 4% paraformaldehyde (Gibco, Germany) and embedded in low melt agar (Sigma, Germany). In the direct cocultured group, both islet and MSCs were fixed and separated from the plate with a scraper. After preparing 5 μm sections and deparaffinization, the slides were subjected to ICC using primary antibodies against Bcl-2 (Abcam, France, #ab115807), Bax (Abcam, France, #ab69643), active caspase-3 (Abcam, France, #ab32042), HIF-1α (Medaysis, USA, #RM0374), and p53 (Dako, USA, #M7001). HRP-secondary antibody (Abcam, France, #ab6717) was used for detection after overnight incubation with primary antibodies. Positive cells for protein expression appeared with 3,3′-diaminobenzidine (Sigma, Germany, #D12384) staining and counterstained by hematoxylin (Sigma, Germany, #H3136). The protein expression rate was calculated by the H-score method using the following formula: H − score = 1 × (%mild staining) + 2 × (%moderate staining) + 3 × (%strong staining) [3 (link), 28 (link)].

To assess apoptosis in the islets, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using Click-iT® Plus TUNEL Assay Kit (Life Technologies, France, #C10617) based on the manufacturer's protocol. After 5 days of coculture, the islets in all groups were fixed in 4% paraformaldehyde (Sigma, Germany, #P6148) and embedded in low melt agar (Sigma, Germany, #A1296). For the direct cocultured group, both islet and MSCs were fixed and separated from the plate with a scraper. After preparing 5 μm sections and deparaffinization, TUNEL assay was performed. Next, DAPI (Sigma, Germany, #D9542) solution was used for nucleus staining. Imaging was done by fluorescent microscopy. The percentage of apoptotic islets was calculated based on the ratio between the TUNEL-positive cells to the nucleuses [26 (link)].