Contour plus

Measurements of glucose changes over time were performed in the same experimental design as described above (see Cell proliferation assay) using a blood glucometer (Contour® plus ONE ref. 84,656,348, Ascensia Diabetes Care AG) and dedicated strips (Contour® plus ref. 84,581,896, Ascensia Diabetes Care AG). One drop of cell culture supernatant of F-IEX-treated HT-29 and HCT116 was transferred on top of a strip and readout was noted. Measurements were made at three time points (0 h, 48 h, and 72 h). All measurements were performed in triplicate.
To determine lactate levels, HT-29 and HCT116 cells were seeded on a 96-well plate and treated with F > 50 kDa and F-IEX fractions as previously described (see Cell proliferation assay). Lactate concentration was measured after 72 h of treatment using a portable lactate sensor (Lactate Scout+, SensLab GmbH) with dedicated strips (Lactate Scout Sensors ref. 7023-3405-0846, SensLab GmbH). All measurements were performed in triplicate.

Whole blood glucose levels were measured from the tail veins using a Contour Plus glucometer (Ascensia Diabetes Care, Basel, Switzerland). Blood serum was isolated using BD Microtainer tube with serum separator (365967, Becton Dickinson, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Serum insulin was determined by Ultra Sensitive Mouse Insulin ELISA kit (90080, Crystal Chem, Downers Grove, IL, USA). Homeostatis model assessment of insulin resistance (HOMA-IR) was calculated as fasting blood glucose (mmol/L) × fasting insulin (mU/L)/22.5 [15 (link)].

An intraperitoneal (i.p.) glucose tolerance test (GTT) was performed several days before the termination of the 74-day experiment. Animals were fasted for 6 h, and a glucose solution (1 g glucose/kg) was injected i.p. Blood glucose was measured by a portable glucometer (Contour plus, Ascensia Diabetes Care Holdings AG, Switzerland) in blood samples drawn by a needle puncture from a tail vein before and at 15, 30, 60, and 120 min post-glucose injection.
Fasting glucose levels were measured on a portable glucometer and assayed at the last day of the long-term experiment, in animals that were fasted for 12 h. The rats were euthanized by CO₂ inhalation at the end of this experiment, and blood was collected by cardiac puncture. Serum was separated by centrifugation at 1,500 RPM (239^*g) in a Rotina 46R centrifuge (Hettich Zentrifugen, Apeldoorn, the Netherlands) for 10 min at 4°C and stored at −70°C. Chemical analysis of the samples was performed by American Medical Laboratories, Israel (AML), and the results were compared to the control values supplied by AML. Serum levels of insulin-like growth factor-I (IGF-I), were determined using a commercial kit according to the manufacturer's recommendations (Quantikine Mouse/Rat IGF-I assay kit, detection limit 8.4 pg/ml [cat. no. MG100, R&D Systems, Minneapolis, MN, USA]).