1. Nonobese-diabetic mice can be obtained from two suppliers in the United States, The Jackson Laboratory or Taconics. If the studies involve evaluation of spontaneous diabetes incidence, mice should be older than 4-6 wk of age prior to shipping the mice. Shipping younger mice can reduce the spontaneous incidence of diabetes. We allow the mice to acclimate to our facility for 4 wk before initiating any experiments.

2. Occasionally, a mouse will have a transient hyperglycemia, so it is important to get at least two blood glucose measurements.

3. It is believed that the cyclophosphamide selective destroys suppressor cells, leaving pathogenic T-cells unopposed to accelerate the onset of diabetes (5).

4. This technique is used when larger samples of blood need to be drawn.

5. For younger mice to avoid hypoglycemia, this dose may have to be reduced by 50% until their body weight reaches 22-25 g.

6. To maintain a foundation colony, breed the mice only by brother-sister matings. Careful pedigree records will have to be maintained to keep the foundation colony. If your studies involve young adult mice, when you consider the per-diem costs, it is usually more cost-effective to purchase the mice from the suppliers. If the NOD mice will be used for studies of neonatal mice, it is sometimes necessary to breed your own mice. Another consideration is that if the mice are shipped at a very young age from the suppliers, we find that the spontaneous rate of diabetes is lower. Thus, for experiments involving very young mice, it is necessary to breed your own mice.

7. We have also readily obtained excellent results using the RIA kits from Linco (St. Louis, MO). To avoid radioactivity in recent years, we have switch to the enzyme-linked immunoassays (ALPCO).

8. Sera can be stored at 2-8°C up to 24 h or can be frozen at -20°C. Avoid repeated freezing and thawing of samples.

9. The ALPCO Rat Insulin ELISA is a solid-phase two-site enzyme immunoassay.

10. Most of the reagents used in this assay are delivered with the kit.

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