Insulin tolerance test and random fed insulin test protocol

Mice are maintained in a normal light/dark cycle according to the standard protocols of the Joslin Diabetes Center Animal Care and Use Committee.

Mice are tested with age matched or litter mate controlls and typically at least 12 mice per group are required.

The day of the test mice are prepared for the insulin tolerance test: animals are weighed, the tail is nicked with a fresh razor blade by a horizontal cut of the very end, ~35 to 50 microliters of blood is very gently massaged from the tail to an eppendorph tube which is immediately placed on ice, baseline blood glucose is measured by the glucose oxidase method using a Glucometer Elite glucometer, and 0.75 units per kg body weight of diluted Regular Human Insulin (Lily) is drawn up in a Beckton Dickenson D 29 gage 1/2" insulin syringe (insulin is diluted to 1:1000 (0.1 inits/ml) with regular insulin diluent) (3/4 of a unit of 1:1000 insulin for every gram of body weight).  Animals are transfered to individually labeled 1000cc cardboard soup cups with the lid liners removed.

When all mice have been prepared the test is begun.  Inulin is injected into the intraperitoneal cavity.  At 15, 30, and 60 minutes blood glucose is sampled from the tail of each mouse by gently massaging a small drop of blood onto the glucometer strip. Insulin injections and blood glucose sampling is timed to take approximately the same amount of time per animal (i.e. 25 animals are injected in 12 minutes and blood glucose sampling of those same 25 animals should also take about 12 minutes) so that the sample times are accurate for each animal.

Random fed immunoreactive insulin levels: whole blood samples are spun in a refrigerated microfuge at 14,000 rpm for 10 minutes and transfered to a clean tube. 6 microliters of serum is tested using an ELISA assay (Crystal Chem) with mouse insulin as a standard according to the standard protocol.