Krebs buffer

Pancreatic islets were isolated from BALB/c male mice. Islets were pretreated for 1 h with 20% conditioned medium from skin fibroblasts or SphCs, followed by exposure to a proinflammatory cytokine cocktail (8 ng/mL TNF-α, 4 ng/mL IFN-γ, and 0.8 ng/mL IL-1β; PeproTech, USA). After 24 h, islets were labeled with 1 μM Newport Green, 1.5 µM propidium iodide (PI), and 1 µg/mL Hoechst 33342. The death rate of each islet was considered the PI-positive area (nonviable cells), which was divided by the total area of the islet. Quantification was evaluated by ImageJ software (NIH, USA).
Five islets/well were cultured for 24 h with 20% conditioned medium from skin fibroblasts or SphCs. Islets were incubated with Krebs buffer (Sigma-Aldrich, USA) supplemented with 0.2% BSA and 5.6 mM glucose for 30 min at 37°C. The buffer was replaced with Krebs buffer containing 0.2% BSA/2.8 mM glucose for 1 h. The supernatant was collected, and islets were incubated for 1 h in Krebs buffer (0.2% BSA/16.7 mM glucose).

NCI-H716 (American Type Culture Collection [ATCC] CCL-251) cells were grown in RPMI (ATCC) medium supplemented with 10% (vol/vol) heat-inactivated newborn calf serum (NBCS; Gibco). Cultures were maintained at a concentration of 2 × 10⁵ to 8 × 10⁵ cells/ml and used at passages 15 to 40 for cell studies. For cell studies, 96-well plates were coated with 100 μl of 10 mg/ml Matrigel (BD Biosciences) for 2 h at room temperature. Following coating, NCI-H716 cells were seeded at a concentration of 1 × 10⁵ cells/well in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (vol/vol) NBCS, as determined by trypan blue staining using a hemocytometer. Two days later, lyophilized bacterial supernatants were resuspended in Krebs buffer (Sigma) containing 0.2% (wt/vol) bovine serum albumin (BSA) and 0.03% (wt/vol) bovine bile and incubated on the NCI-H716 cells at 37°C with 5% CO₂. 4-Phorbol 12 myristate 13-acetate (PMA, 2 μM) was used as a positive control, as it is a potent stimulator of GLP-1 secretion through the activation of protein kinase C (PKC). Following a 2-h incubation, supernatants were collected and analyzed for total GLP-1 levels by ELISA (MilliporeSigma), according to the manufacturer’s protocol. Cell viability was monitored using the PrestoBlue cell viability reagent (Thermo Fisher Scientific), following the manufacturer’s instructions.

Mucoadhesion of Caricol^®-Gastro was investigated using gastric porcine excised mucosa on a modified falling liquid film technique as previously described by Gradauer et al. [39 (link)]. For this, porcine stomach was obtained from freshly sacrificed pigs (age < 6 months; Marcher Fleischwerke, Graz, Austria) and transported in 4 °C Krebs buffer (Sigma-Aldrich^®, Munich, Germany) to the laboratory. The gastric mucosa was cut from the grater curvature into 20–30 cm² pieces, carefully rinsed with Krebs buffer and mounted with the mucosal side up on a semicylindrical Plexiglas^® tube at a 35° angle. The equipment was put on a shaker, agitated at 100 rpm to mimic gastric motility [47 (link)]. A peristaltic pump was used to constantly flush the mucosa with 0.1 M HCl (pump speed of 2 mL/min), simulating the gastric fluid in the stomach. For visualization purpose, 2 g of Caricol^®-Gastro were died with 5 mg of methylene blue and directly applied on the top of the tube to mimic the transport through the esophagus and the gastric fold to the pyloric sphincter. The amount of remaining material was macroscopically investigated over a time period of 30 min and compared to 2 g organic oat I and oat II dyed with 5 mg of methylene blue and 2 g MQ-water dyed with 5 mg of methylene blue (control).

Human islets (~20–50 islets) and hiPSC-derived cells (1~2 × 10⁶) were rinsed twice with Krebs buffer (Sigma-Aldrich) and pre-incubated in Krebs buffer for 1 h to remove residual insulin. Cells were then incubated in Krebs buffer containing 2.8 mM glucose for 1 h, followed by incubation in Krebs buffer containing 16.7 mM glucose. Supernatant samples were collected after each incubation period, centrifuged to remove cell debris and frozen at −80 °C. Human insulin and C-peptide released into the medium were measured by an ELISA kit (R&D) according to the manufacturer’s instructions. Total protein contents were measured using a BCA protein assay kit (Pierce Biotechnology, USA), and insulin/C-peptide levels were normalized to protein levels. Written informed consent was obtained from all participants, and the use of human samples was approved by the Ethics Committees of the Second Clinical Medical College of Jinan University.

l-[³H]Arginine uptake was measured in control or y⁺LAT2 silenced astrocytes, treated or not with 5 mM ammonium chloride. Cells were washed three times with Krebs buffer (Sigma-Aldrich, St. Louis, MO, USA). Basal arginine concentration in the cells was adjusted to 100 μmolar concentration by incubation in Krebs buffer containing 100 μM arginine for 10 min in 37 °C. Thereafter the solution was replaced by incubation mixtures containing Krebs buffer with 0.1 μCi/mL l-[³H]Arginine (specific radioactivity 37 MBq/mL; Hartmann Analytic) unlabeled arginine at 100 μmol/L and ADMA in the concentrations: 5 μM, 50 μM, 0.5 mM, 1 mM, and the incubation was continued for 20 min. Cells were then washed three times in ice-cold Krebs buffer, lysed in 0.5 mL 1M NaOH and protein content was determined. The radioactivity of cell lysates was measured in a Wallac 1409 (Perkin–Elmer, Turku, Finland) liquid scintillation counter.

Pre-vascularized pancreatic islet spheroids were individually placed in a 96 well plate with ultra-low attachment surface, and incubated at 37 °C, 95% O2, 5% CO2 for 1 h in 60 µl KREBS buffer (Sigma-Aldrich, cat. no. K4002), 1% BSA, 2.8 mM glucose (Gibco, cat. no. A2494001) as pre-incubation. Thereafter, islets were incubated for 1 h with 2.8 mM glucose solution (low glucose solution) and for 1 h with 16.7 mM glucose solution (high glucose solution). After each incubation step, supernatants were collected and stored at −80 °C. Insulin concentration in each collected supernatant was measured using STELLUX Chemiluminescence Human Insulin ELISA (ALPCO, cat. no. 80-INSHU-CH01). Similar protocol was performed for on-chip experiments, following a protocol previously described^{42 (link)}. Each sample was measured in duplicate. A stimulation index was obtained by calculating the ratio of insulin measured between high and low glucose stimulation, [high glucose solution]/[low glucose solution].