Hanks balanced salt solution (hbss)

Respiratory burst was measured using chemiluminescence (CL). The assay was performed in Nunc-Immuno™ MicroWell™ 96-well polystyrene plates (Sigma-Aldrich). Purified CD14⁺ monocytes were seeded into the plate on the day of isolation at a concentration of 1 × 10⁵ cells per well in 0.2 mL of complete medium and cultured as described above. The old medium was removed immediately before measurement, the cells were washed with HBSS without calcium and magnesium (Lonza), and HBSS with calcium and magnesium was then added. Luminol derivative L-012 (Wako Chemicals GmbH, Neuss, Germany) was added to amplify the CL induced by the respiratory burst of stimulated cells. L-012 was diluted in HBSS to a final concentration of 0.15 mmol/L. CL was measured in cells stimulated by phorbol myristate acetate at a final concentration of 0.5 µg/mL (Sigma-Aldrich) and in non-stimulated cells (spontaneous chemiluminescence). The final volume of each well was 200 µL, and culture triplicates were included for all stimulations. Chemiluminescence was measured using a multi-detection microplate reader Synergy H1 (BioTek, Winooski, VT, USA) in kinetic mode for 2 h. The results were expressed as integrals of chemiluminescence intensity.

Pancreata from 12-week-old male C57BL/6NJR mice (Janvier, Saint Berthewin Cedex, France) or 12-week-old male C57BL/6NTAC mice (Taconic, Lille Skensved, Denmark) were inflated with Liberase (Roche, Hvidovre, Denmark), excised and incubated at 37 °C to allow digestion. Digestion was halted by adding Hanks’ balanced salt solution (HBSS; Lonza, Basel, Switzerland) supplemented with bovine serum albumin (BSA; Sigma, Soeborg, Denmark). Islets were handpicked and cultured for 1 day in 10 ml RPMI 1640 medium (Lonza) with 11 mmol/l d-Glucose supplemented with 10% fetal bovine serum (FBS; Biosera, Herlev, Denmark) and 1% penicillin/streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin) (P/S; Gibco, Life Technologies, Roskilde, Denmark). Thereafter islets were cultured for 10 days at 37 C° in a humidified atmosphere with 5% CO₂ in 10 ml RPMI 1640, 2% human serum (HS; Lonza), and 1% P/S in the absence or presence of 50 ng/ml recombinant human BMP-2 (R&D Systems, Life Technologies). The medium was changed every fifth day. For GSIS, apoptosis and proliferation analysis, islets from individual mice were used. For RNA isolation, Proteomic and ChIP-seq analysis, pools of islets from several mice were used. All animal experiments were approved by the local ethics committee, and animals were housed according to the Principals of Laboratory Care.

In order to analyze the phagocytic activity and phagolysosome colocalization of PBMC-DM cells by flow cytometry, fungal cells were labeled with Alexa Fluor 647 succinimidyl ester and pHrodo red succinimidyl ester (Invitrogen) as follows. First, 22 µl Na₂CO₃ (1 M, pH 10), 4 µl Alexa Fluor 647 (1 mg/ml in DMSO), and 4 µl pHrodo red (100 µg/ml in DMSO) were added to 200-µl fungal cell suspensions in Hanks’ balanced salt solution (HBSS) (Lonza) and incubated for 1 h in the dark at room temperature. pHrodo red stains the fungal cell wall and emits fluorescent light only in a highly acidic environment such as the phagolysosome. Fungal cells were then washed four times with HBSS, and cell concentrations were adjusted to the appropriate concentration. PBMC-DM cells were infected with the labeled fungal cells at a 1:5 ratio in 12-well cell culture plates and incubated for 2 h (5% CO₂, 37°C, 100% relative humidity). After the incubation, extracellular fungal cells were removed by washing the wells with PBS. Macrophages were harvested from the wells with trypsin (5 mg/ml; Sigma-Aldrich). Samples were measured in PBS with a FlowSight instrument (Amnis), and data were analyzed with the IDEAS 6.2 software.

Bone marrow cells were isolated according to the procedure outlined in the Stem Cell Technologies Technical Manual version 3.1.1(http://www.stemcell.com/). Briefly, each mouse was sacrificed and 70% isopropyl alcohol was immediately used to wet the ventral fur to avoid contamination at site of dissection. Both femurs were collected sterilely and placed and held on ice in Hanks' Balanced Salt Solution (HBSS) purchased from Lonza (Walkersville, MD). To extract cells, femurs were placed in petri dish containing cold sterile RPMI 1640 Medium supplemented with 2% Fetal Bovine Serum (FBS). The ends of the femurs were trimmed to expose interior marrow shaft. Using a 1 cc syringe with a 25 gauge needle, approximately 1 ml cold sterile medium was flushed through the femur several times to release cells into a petri dish. The medium containing cells from both femurs was immediately transferred to a 15 ml culture tube and placed on ice until needed. Cells were washed via centrifugation at 4°C, 400 × g for 10 min and were resuspended in RPMI media (as above) for culturing or flow cytometry analysis as discussed below. Cell viability was determined by Acridine Orange/Propidium Iodide (AO/PI) staining and counting using the Nexcellom Cellometer 2000.