Incubation of Isolated Muscles

1. Epitrochlearis muscles are removed under anesthesia from rats weighing approx 100 g (5,8). These muscles are ideally suited for incubation because they are thin and flat and give good glucose-transport responses to contraction (9). Alternatively, soleus muscles can be used for these experiments; however, these muscles do not respond as well to contractions and must be split prior to use in incubations, due to their thickness (8,10).

2. The excised muscles are rinsed in 0.9% saline and placed in 2 mL of preincubation media (KHB supplemented with 0.1% BSA, 32 mM mannitol, and 8 mM D-glu-cose) in 25-mL Erlenmeyer flasks in a 30°C water bath. The flasks are continuously gassed with 95% O2/5% CO2 and shaken at 1 cycle/s for 1 h.

3. Muscles are next transferred to the stimulation apparatus containing incubation media (2 mL; same as preincubation solution) as outlined in Subheading 3.2.

Fig. 1. Stimulators used for muscle stimulation. Body of device is made from Delrin plastic and is designed to fit into a Falcon 2059 (17 X 100) tube. Tube at top extends to bottom of device to allow gassing of media. Phono plug-type connector on top of device is attached to wires that run down the back of device and are attached to platinum wires with screws at right. Muscles are pinned to rubber pads at right and lie in between electrodes.

Fig. 1. Stimulators used for muscle stimulation. Body of device is made from Delrin plastic and is designed to fit into a Falcon 2059 (17 X 100) tube. Tube at top extends to bottom of device to allow gassing of media. Phono plug-type connector on top of device is attached to wires that run down the back of device and are attached to platinum wires with screws at right. Muscles are pinned to rubber pads at right and lie in between electrodes.

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