Earle s balanced salts solution ebss

To examine the effects of glutamate on glucose uptake and determine the optimal concentration of glutamate to stimulate maximal glucose uptake, we first treated cells with increasing doses of glutamate to observe the relationship between glutamate exposure and glucose uptake in skeletal muscle.
Prior to treatments, fully differentiated L6 myotubes were washed 2-3x in DPBS before changing media to serum-free DMEM for 4 hrs followed by 1 hr incubation in amino acid-free Earle's Balanced Salts Solution (EBSS; Sigma-Aldrich). The purpose of this protocol was to wash out the effects of insulin and other compounds in the serum, and the high concentration of amino acids in DMEM, that can effect glucose uptake and mask any effects of the treatments. Cells were then washed at least 2 more times treated with L-glutamic acid (Sigma-Aldrich) in EBSS for 1 hr. The concentrations of glutamate used for the treatments are representative of: i) typical human serum fasting concentration (50µM); ii) approximate human serum glutamate concentrations following a 150mg/kg dose (500µM) (Di Sebastiano et al., 2013) (link); and iii) + iv) supra-physiological glutamate doses to entice a maximal response to the treatment. Immediately following the treatments, cells were washed 2-3x in DPBS and a glucose uptake assay was performed.