Omni th tissue homogeniser

Sarkosyl-insoluble tau was isolated from the brainstem of 5- to 6-month-old rats. Frozen brain samples of the studied rats were homogenised in 10 vol of ice-cold extraction buffer (SL buffer: 20 mM Tris, pH 7.4; 800 mM NaCl; 1 mM ethylene glycol tetraacetic acid), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% β-mercaptoethanol, 10% sucrose, 1 mM Na₃VO₄, 20 mM NaF, supplemented with EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) using an OMNI TH tissue homogeniser (OMNI International, Kennesaw, GA, USA). After 5-minute incubation on ice, the homogenates were cleared by centrifugation at 20,000 × g for 20 minutes at 4 °C. Solid sarkosyl (N-lauroyl sarcosine, Na-salt; Sigma-Aldrich) was added to the supernatant to achieve 1% concentration and stirred for 1 h. Thereafter it was centrifuged at 100,000 × g for 1.5 h at room temperature. The supernatant was collected, and pellets were gently rinsed with 1 ml of the SL buffer and centrifuged for 20 minutes at room temperature. The pellets were dissolved in sodium dodecyl sulphate (SDS) sample loading buffer.

The expression of inducible isoform of nitric oxide synthase (iNOS) in colonic epithelial cells in response to pro-inflammatory stimuli [33 (link)] was determined in PC and DC specimens using a Nitic Oxide Synthase Activity Assay kit (ab211084, Fluorometric, Abcam^®, Cambridge, UK). Snap frozen proximal and distal colonic tissues (n = 3) were washed in in cold PBS and resuspended in 200 μL NOS assay buffer then homogenised by 10–15 passes of an Omni TH tissue homogeniser (Omni International, Tulsa, OK, USA). The homogenate was then centrifuged at 10,000× g (10 min, 4 °C) and the supernatant then underwent iNOS activity assay activity assay as per the manufacturer’s instructions. The amount of protein in the lysate was determined using DC™ Protein Assay (Bio-Rad Laboratories, Australia). The results are expressed as iNOS activity mU/mg.

The extent of the inflammatory cell invasion in the colon was examined by the assessment of myeloperoxidase (MPO) activity [24 ]. Weighed and snap frozen PC and DC specimens (n = 3) were analysed for MPO activity using a Myeloperoxidase Activity Assay kit (ab105136, colorimetric, Abcam^®, Cambridge, UK). Briefly, frozen tissue after washing in cold PBS, was resuspended in MPO assay buffer, before homogenisation with 10–15 passes using an Omni TH tissue homogeniser (Omni International, US) with 10–15 passes. The homogenate was then centrifuged at 13,000× g (10 min) and the supernatant assayed for MPO activity as per the manufacturer’s instructions. The values are expressed as MPO activity units/g tissue.