Mouse Insulin Immunoassay of Plasma Acquired During Glucose Tolerance Testing in Wild Type or Transgenic Mice

An example of insulin secretion during glucose tolerance testing in mice is shown in Fig. 5.

1. Label duplicate plain 12 X 75-mm tubes in the sequence: [T], [N], [R], and [4-n].

2. Pipet 0.1 mL RIA buffer into tubes [N] and [R]. Pipet 0.1 mL of each standard solution into tubes [4-9], beginning with 5 pg/mL into tube [4].

3. Pipet 0.1 mL each diluted plasma sample into the remaining tubes, beginning with the first sample into tube [10].

4. Add 0.1 mL diluted primary antibody (repeating pipet) to all tubes except [T] and [N]. Add 0.1 mL NGPS buffer to tubes [N]. Mix, seal the tubes in the rack as in the solid phase assay and incubate for 72 h at 4°C.

5. Add 0.1 mL of diluted 125I-insulin (repeating pipet) to all tubes, reseal the bags, and continue the incubation for 24 h at 4°C.

6. Add 0.1 mL diluted secondary antibody to all tubes except [T]. Mix, reseal, and incubate for 3 h at 4°C.

7. Add 1.6 mL ice-cold separation buffer (repeating pipet) to all tubes except [T]. Add separation buffer to only those tubes that can be centrifuged immediately. Keep the remainder covered and at 4°C. Without mixing and without delay, transfer the tubes to the precooled centrifuge adapters and centrifuge for 30 min at 6000g at 4°C.

Fig. 5. Impaired glucose tolerance in mice heterozygous for PDX-1. Fasted mice received glucose by intraperitoneal injection (2 g/kg body weight). Blood samples collected from retro-orbital bleeds were immediately analyzed for glucose. Plasma was collected from the remainder of the blood sample for insulin RIA. Open circles: wildtype mouse; filled circles: PDX-1 heterozygote mouse.

Fig. 5. Impaired glucose tolerance in mice heterozygous for PDX-1. Fasted mice received glucose by intraperitoneal injection (2 g/kg body weight). Blood samples collected from retro-orbital bleeds were immediately analyzed for glucose. Plasma was collected from the remainder of the blood sample for insulin RIA. Open circles: wildtype mouse; filled circles: PDX-1 heterozygote mouse.

8. Without delay and without removing the tubes from the centrifuge adapters, remove the adapters from the swinging buckets, place the wire gauze over the tubes, grip the wire-adapter assembly with both hands (fingers on the outer edge of the wire and thumbs underneath the adapter), and invert the assembly for 10 s. Return the assembly to the horizontal position, replace the wire with the paper toweling pad, grip the pad-adapter assembly as before, guide the spindle of the first adapter into the 27-hole of the 6 X 12-place test tube rack (spindle of the second adapter into the 34-hole), allowing the weight of the adapter to settle the tubes onto the pad for blotting (see Note 18).

9. After blotting for 1 min, transfer the tubes to gamma counter racks for counting (5 min/tube) (see Notes 19-21).

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