Liberase tl grade

Mice were killed using CO₂ suffocation after which the spleens were harvested. Spleens were digested with digestion buffer (1.5 WU/mL liberase TL grade, 100 Units/mL recombinant DNAse I, both Roche, Basel, Switzerland) for 30 min at 37 °C and meshed through a 40 µm filter (BD Biosciences) to prepare a single cell solution using PBS/0.5 mM EDTA wash buffer. The red blood cells were lysed using an isotonic ammonium chloride buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) for 5–10 min on ice, washed 1x with PBS, after which the cells were counted and resuspended in high glucose DMEM (Thermo Fisher scientific) supplemented with 10% FCS. 5x10⁵ splenocytes were added per well in a U-bottom 96-wells plate (Corning). Total splenocytes were stimulated with 1 µg LTA-SA or the different S. suis strains at an MOI of 0.2. After 2 h, the supernatant was collected (by centrifugation at 700x g for 2 min) and the cells were resuspended in 100 µL fresh medium supplemented with 200 µg/mL gentamycin and left for an additional 22 h. After 24 h, medium was collected and stored at −20 °C for cytokine measurements.

All experimental procedures were approved by the Animal Ethics Committee of the University of Adelaide and conform to the guidelines set out by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Primary pancreatic islets were isolated from 6- to 12-week-old male C57B6 mice (University of Adelaide Laboratory Animal Services, Adelaide, SA, Australia). Briefly, 3 mL cold M199 medium (Sigma–Aldrich) containing 0.67 mg collagenase (Liberase TL grade; Roche Diagnostics, GmbH, Germany) per pancreas was infused into the pancreatic duct in situ. The pancreas was removed and digested at 37 °C for 14–16 min. Islets were purified on a Ficoll gradient (GE Healthcare, Amersham, UK). Following extensive washing, islets were grown (37 °C, 5% CO₂) in RPMI 1640 medium ( Sigma–Aldrich) supplemented with 2.5 mM GlutaMax, 100 U/mL streptomycin, 100 µg/mL penicillin, and 10% fetal calf serum for up to 2 days. Primary mouse islets (2 × 10² islets/mL) were seeded onto array and cultured in contact with arrays at 37 °C and 5% CO₂. Non-bound islets were rinsed off after 16 h by washing with prewarmed RPMI. The cells were subsequently incubated in fresh CMRL 1066 medium including 15% FBS at 37 °C and 5% CO₂ for 2 days.