Htrf assay

After two-round infection with virus, human islets were washed extensively with Dulbecco’s phosphate buffered saline (DPBS) and then equilibrated for 30 min at 37 °C incubator with Krebs Ringer Buffer (KRB) + 2.8 mM glucose. Cells were stimulated with 16.7 mM glucose for 1.5 h at 37 °C. Supernatant/cells were collected and frozen for subsequent ELISA and protein assay. Insulin was detected with HTRF assay (Cisbio) kits or ELISA (Crystal Chem) and performed on the PHERAstar microplate reader.

Islets isolated from individual mice at 1 week were cultured in RPMI 1640 medium with 10 mmol/l glucose and 10% fetal bovine serum for 2 days. Islet function was evaluated by perifusion as previously [24 (link)], with 3 mmol/l basal glucose (ramp of 0.625 mmol l⁻¹ min⁻¹), followed by 30 mmol/l KCl stimulation at completion. Insulin secretion was measured by HTRF assay (Cisbio, Bedford, MA, USA). Cytosolic calcium was measured as described previously [24 (link)]. Fura-2AM (Life Technologies) was used as a calcium indicator and was measured with a Zeiss AxioVision microscope (Carl Zeiss, Thornwood, NY, USA).

Islet isolation by collagenase injection and pancreatic distension was performed as described [43] (link). Islets were cultured for 24–36 h in RPMI medium supplemented with 10% foetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Hormone release was measured during 30 or 60 min (insulin or glucagon, respectively) static incubation of batches of six (insulin) or 20 (glucagon) islets in 0.5 mL modified Kreb's Ringer medium comprising (mM): 120 NaCl, 4.8 KCl, 24 NaHCO₃, 0.5 Na₂HPO₄, 5 HEPES, 2.5 CaCl₂, 1.2 MgCl₂ and 5 d-glucose, pH 7.4, at 37 °C. Secreted and total hormone levels were measured by homogeneous time-resolved fluorescence-based (HTRF) assay (Cisbio).

IP₃ was measured using IP₁ as a surrogate [45 (link)] with an HTRF assay (Cisbio). The assays were performed in triplicate in 96-well plates, and the signal was quantified on an Infinite M1000 PRO plate reader (Tecan).

Compound activity on endogenous NOD1 and NOD2 receptors was determined by inhibition of Tri-DAP or MDP-stimulated IL-8 release using HCT116 human colon carcinoma cells, maintained in DMEM high glucose medium supplemented with 25 mM HEPES and 10% FBS. For assay, cells were seeded into 96 well plates (90,000 cells/well) in DMEM containing 1% FBS and pre-incubated with compounds for 1 hour before addition of either 25 µg/mL Tri-DAP or 1 µg/mL MDP. The level of IL-8 released into medium after 24-hour incubation was determined by HTRF assay (Cisbio) as described above and fluorescence measured on an Envision model 2102 multilabel plate reader (Perkin Elmer). The activity of compounds against NOD1-induced type I interferon response was determined by inhibition of Tri-DAP stimulated IP-10 secretion in HT-29 cells, maintained in the same medium as for HCT116 cells. The HT-29 cells were seeded into 96 well plates (60,000 cells/well) in DMEM with 1% FBS, pre-incubated with compounds for 1 hour and stimulated for 24 hours with 50 µg/mL Tri-DAP. The concentration of IL-8 released into the medium was measured by HTRF as above, IP-10 was measured using the IP-10/CXCL10 Quantikine ELISA kit (R&D Systems), and IFNβ was measured using the MA6000 human IFN-β tissue culture kit (Meso Scale Discovery).