Tissue chopper

Organotypic cerebellum brain slice cultures were prepared according to previously published protocols [35 (link),36 (link),37 (link),38 (link)]. In brief, cerebella were derived from P10–P12 mice and separated from the rest of the brain. The cerebellum was dissected in ice-cold minimum essential media (MEM) supplemented with Glutamax (Gibco) until taken for cutting sagittal slices of 300 µm thickness using a McIlwain Tissue chopper. The separated slices were transferred onto humidified sterile culture membrane inserts with pore sizes of 0.4 µm (Merck Millipore, Watford, UK). Tissue slices on membrane inserts were cultured on a liquid layer of medium containing 50% MEM media (Gibco), 25% HS, 25% Earle’s Balanced Salt Solution (EBSS) (Gibco) supplemented with 130 mM D-glucose (Sigma) and 1% P/S. The cultures were first maintained at 37 °C in 5% CO₂ for 24 h prior to being used for in vitro experiments. After 24 h, the medium bathing the tissue was replaced with serum-free medium, into which treatments were added including vitamin K1 (100 µM) or warfarin (25 µM). After 72 h incubation, the culture medium was analysed for released Gas6 protein by ELISA assays for both total mouse Gas6 and Gla-Gas6 (below).

Single cell suspensions of whole prostates were derived using a tissue-chopper (McIlwain) followed by 2 hours incubation in DMEM (Gibco) containing 1 mg/ml collagenase (Roche) plus hyaluronidase 5000 u/ml (Sigma) at 37°C. Samples were filtered, spun down and re-suspended in FACSbuffer (PBS +0.5% BSA), stained with antibodies against CD45, CD11b, CD3, CD19, B220, F4/80, Ly6G (APC, FITC and PE, all Ebioscience) and analyzed (whole single cell suspension sample for absolute numbers) on a FACS Fortessa (BD Biosciences) using FlowJo software (V10).

Male Zucker diabetic fatty rats (ZDF, n = 8) and control rats (Lean, n = 8) were obtained from Charles River Laboratories at 6 weeks of age with an average weight of 266 g for ZDF and 203 g for Lean. The animals were fed water and chow ad libitum throughout the experiment till they reached 17 weeks of age. Blood samples were taken every two weeks from week seven to week seventeen. Glucose and HbA_1c levels were measured by commercial kits (Roche). At 17 weeks of age, rats were anesthetized by 2.5% isoflurane and sacrificed by exsanguination. Brains were removed and either snap frozen in liquid nitrogen or cut into 200 μm thick slices using a McIlwain tissue chopper and placed in brain nutrition medium/neurobasal medium (Gibco BRL, Cat. No. 21103–049) for in vitro experiments. The concentration of glucose in frontal brain homogenates was measured using Accu-Check Aviva (Roche diagnosis, Mannheim, Germany) in which a drop of homogenized brain (n ≥ 5 samples in each group) was placed on the glucose test strip and read by the instrument. All experiments were approved by the Animal Care Committee of the University Medical Center Groningen (Dec 6032B).