Diastix reagent strips

Urinary glucose was monitored twice weekly from 12 weeks of age using Diastix Reagent Strips (Bayer, Pymble, NSW, Australia). Glycosuric mice were tested for blood glucose using Accu-Chek Go System meters (Roche, Dee Why, NSW, Australia). Mice were classified as diabetic and killed following two consecutive blood glucose readings >15 mm. For analysis of insulitis, pancreata were collected, fixed in formalin and embedded for routine histology. Insulitis was graded in hematoxylin and eosin (H&E) stained sections on a scale of 0–4 as described, in at least 25 islets.^{45 (link)} Data are expressed as the proportion of islets of the total with each score.

Mice were tested once a week for elevated urinary glucose using Diastix reagent strips (Bayer diagnostics). Mice with a positive glycosuria reading (>110 mmol/L) and confirmed by a positive blood glucose reading (>13.0 mmol/L), using Advantage II Glucose Strips (Roche), were diagnosed as diabetic. Pairwise comparisons of diabetes incidence curves were performed using the log-rank test. When more than two comparisons were made between diabetes incidence curves, the P values were adjusted using the Holm method for multiple testing [28 (link)].

C57Bl/6J, NOD-SCID, and NOD mice were housed and inbred in the animal facility of Katholieke Universiteit Leuven (KU Leuven, Leuven, Belgium) as previously described (27 (link)). All animal procedures were performed in accordance with the NIH guidelines for the care and use of laboratory animals and protocols were approved by the Ethics Committees of the KU Leuven. NOD female mice used in this study were screened for the onset of diabetes by evaluating urine glucose levels (Diastix Reagent Strips; Bayer, Leverkusen, Germany) and venous blood glucose levels (Accu-Chek; Roche Diagnostics, Vilvoorde, Belgium). Mice were diagnosed as diabetic when they had glycosuria and two consecutive blood glucose measurements exceeding 200 mg/mL. Pancreatic sections used for histological analysis were collected from 8-week-old C57Bl/6J (n = 4), 15- to 20-week-old C57Bl/6J (n = 3), 2- to 3-week-old NOD (Non-Obese Diabetic)-SCID (severe combined immunodeficient) (n = 4), 20-week-old NOD-SCID (n = 3), 20- to 22-week-old normoglycaemic NOD (n = 4) and 12- to 21-week-old new-onset diabetic NOD mice (n = 4).

Mice were placed in a clean, sawdust-free cage. Most mice urinated spontaneously, but where necessary, gentle handling was used to induce micturition. The urine was tested with Diastix reagent strips (Bayer plc, Newbury, UK), which enables a semi-quantitative analysis of glycosuria. The presence of glycosuria was regarded as indicative of diabetic status. Mice were tested at 12 and 16 weeks of age.

Glucosuria was monitored weekly using Diastix reagent strips (Bayer, Elkhart, IN). Mice were considered diabetic after two consecutive positive tests, and the date of the first positive test was recorded as the time of onset of disease.