Mouse Insulin Immunoassay of Samples Acquired During Perfusion of the Mouse Pancreas In Situ

An example of insulin secretion data from the in siiw-perfused mouse pancreas is shown in Fig. 4.

1. Label duplicate plain tubes in the sequence: [T] (total counts) and [N] (nonspecific binding).

2. Label duplicate antibody-coated tubes in the sequence: [R] (reference or maximum binding), [4-10] (reserved for standard insulin), and [11-n] (n-number of samples). Put tubes in wire racks.

4. Pipet 0.1 mL each insulin standard solution into tubes [4-10] beginning with 0.25 ng/mL into tube [4].

5. Pipet 0.1 mL each sample (neat or appropriately diluted in RIA buffer) into the remaining tubes beginning with sample 1 into tube [11].

6. Add 0.9 mL 125I-insulin (repeating pipet) to all tubes. Mix and seal each rack of tubes in a polyethylene bag (see Note 11).

8. Transfer the tubes (except [T]) to the foam racks. Without removing the tubes from the foam racks, decant the non-antibody-bound counts, leaving the antibody-bound

200 150

I 20

Fig. 4. Insulin secretory response of the in sim-perfused pancreas. Insulin secretion from the pancreas of a PDX-1 heterozygote mouse (filled circles) and its wild-type littermate (open circles) was analyzed in response to glucose and arginine. The integrated response to 16.7 mM glucose was 81 ng insulin in the PDX-1 (+/+) mouse versus 31 ng insulin in the PDX-1 (+/—) mouse. The integrated response to 20 mM arginine in the presence of 5.6 mM glucose was 381 ng insulin in the PDX-1 (+/+) mouse versus 317 ng insulin in the PDX-1 (+/—) mouse. The integrated response to 16.7 mM glucose + 20 mM arginine was 1264 ng insulin in the PDX-1 (+/+) mouse versus 575 ng insulin in the PDX-1 (+/—) mouse.

counts on the walls of the tubes and blot the tubes on the absorbent surface (see Note 12).

9. Transfer the tubes to appropriate racks for counting in a gamma counter (5 min/tube) (see Notes 13 and 14).

200 150

Insulin Tubes

Fig. 4. Insulin secretory response of the in sim-perfused pancreas. Insulin secretion from the pancreas of a PDX-1 heterozygote mouse (filled circles) and its wild-type littermate (open circles) was analyzed in response to glucose and arginine. The integrated response to 16.7 mM glucose was 81 ng insulin in the PDX-1 (+/+) mouse versus 31 ng insulin in the PDX-1 (+/—) mouse. The integrated response to 20 mM arginine in the presence of 5.6 mM glucose was 381 ng insulin in the PDX-1 (+/+) mouse versus 317 ng insulin in the PDX-1 (+/—) mouse. The integrated response to 16.7 mM glucose + 20 mM arginine was 1264 ng insulin in the PDX-1 (+/+) mouse versus 575 ng insulin in the PDX-1 (+/—) mouse.

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