Trypsin solution

The PC-12 pheochromocytoma cell line derived from rat adrenal medulla was obtained from American Type Culture Collection (Manassas, VA). RPMI-1640, trypsin solution, phosphate-buffered saline, fetal bovine serum (FBS), sodium pyruvate (0.1 M), L-glutamine (0.2 M) and penicillin/streptomycin solution (containing 10,000 units·ml⁻¹ penicillin and 10,000 μg·ml⁻¹ streptomycin) were obtained from Quality Biological (Gaithersburg, MD), horse serum (heat inactivated) was purchased from Biosource (Rockville, MD) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer [1 M, pH 7.4] was obtained from Mediatech, Inc. (Manassas, VA). The PC-12 cells were maintained in RPMI-1640 supplemented with 1 mM HEPES buffer, 10% horse serum, 5% FBS, 1% sodium pyruvate, 1% L-glutamine and 1% penicillin/streptomycin.

(R,R’)-MNF was synthesized as previously described.^{14 (link)} Lactate (purity ≥ 98%), 3-hydroxybutyrate (purity ≥ 98%), carnitine (purity ≥ 98%), p-aminohippuric acid (purity ≥ 98%), human recombinant insulin (dry powder), methanol (HPLC grade), acetonitrile (HPLC grade) and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, Eagle’s Minimum Essential Medium (EMEM), trypsin solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), 100X solutions of sodium pyruvate (100 mM), L-glutamine (200 mM), and penicillin/streptomycin (a mixture of 10,000 units/mL) were obtained from Quality Biological (Gaithersburg, MD, USA).