They were tested routinely for blood glucose levels and considered prediabetic, as their values of serum glucose on two occasions over a 24-h period did not differ significantly from those of control mice (0·9 ± 0·1 g/l, n = 42). NOD mice of 16 weeks of age used in
this study presented a reduced saliva flow rate Cell Cycle inhibitor (>35% reduction) compared with BALB/c control mice. Studies were conducted according to standard protocols of the Animal Care and Use Committee of the School of Exact and Natural Sciences, University of Buenos Aires. Submandibular glands were removed and transferred immediately to ice-cold RPMI-1640, 10% fetal bovine serum (FBS) for acinar cell isolation, as described previously [16]. Acinar cells were washed and seeded on flat-bottomed 24-well microtitre plates (Corning Glass, Corning, NY, USA) and incubated for 2 h at 37°C in a humidified incubator with 5% CO2 to separate immune adherent cells and viability determination [16]. When used, recombinant TNF-α (Promega, Madison, WI, USA) (5–10 ng/ml) was added to acinar cell culture for 3·5 h [reverse transcription–polymerase chain reaction (RT–PCR)] or for 6 h (annexin V staining and immunoblotting). In some experiments, cells were preincubated for 30 min with 100 nm VIP (PolyPeptide Labs, Strasbourg, France) before TNF-α addition in the presence or absence of H89
(1 µm). Macrophages were obtained by washing the peritoneal cavity with ice-cold RPMI-1640, as reported [24,25]. Cells were seeded at 5 × 105 cells/well (Corning Glass), incubated at 37°C for 2 h and washed thoroughly before co-cultures, nuclear Akt inhibitor stiripentol factor (NF)-κB activation or cytokine determination. Macrophages were co-cultured with freshly isolated acini or acini previously induced to apoptosis with TNF-α. Incubations were run at 37°C for the times indicated. VIP (100 nm) was added 30 min before the addition of acini. After incubation, acini were removed and macrophages were
washed with fresh medium. Haematoxylin and eosin (H&E) staining was used for phagocytosis determination [24]. Cells were collected for cytokine expression by quantitative RT–PCR (qRT–PCR) or flow cytometry analysis; nitrite production was determined by the Griess in supernatants, as described previously [24,25]. For flow cytometry, cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 monoclonal antibody for 30 min (eBioscience, San Diego, CA, USA), fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS)–2% FCS, permeabilized with 0·5% saponin (Sigma, St Louis, MO, USA) and incubated with phycoerythrin (PE)-conjugated anti-IL-10 monoclonal antibody (BD) or with the PE-conjugated immunoglobulin (Ig)G1 isotype; 10 000 events were acquired in a fluorescence activated cell sorter (FACS)Aria cytometer® and results analysed using the WinMDI software®.