Cell cultures

            Specimens were maintained and treated with multiple preparative serums approximately four days with limited glucose supply before treatments.  Then the muscle cell line was treated with appropriate substances for the specific experiments (i.e. Caffeine, AICAR, dantrolene) depending on the variable being addressed.  Myotubes were washed to remove the added agents and then the variable of interest was determined.

Isolated Muscle Incubations

            Isolated muscles were obtained from a rat and incubated in the medium for which they would later be tested.

Western Blot Analysis

            Cells homogenized in a buffered solution of isolated proteins underwent electrophoresis (separation by addition of electricity).  Antibodies that bind to the particular protein being tested for were then added.  The separated proteins were added onto special paper and blotted with antibodies specific to that protein.  Any antibody bound proteins were then detectable using chemiluminescence to determine if the protein in question was present in the cell.

Fura 2 epiflourescence digital microscopy

            Cells on cover slips were treated and given an excessive amount of saline solution containing the appropriate additives.  They were then excited to a higher energy state using 340-380nm wavelengths of light.  Upon return to the normal energy state energy is re-emitted (fluoresced) at 510nm.  These data are then collected with a digital camera and saved onto a computer for analysis.  The spectrum created from this test was used to determine intracellular calcium levels.