Glutamate

Diazepam, mitoxantrone HCl, and quetiapine fumarate were obtained as gift sample from Intas Pharmaceuticals, Ahmedabad, India. Sodium valproate was obtained as a gift sample from Chemclone Industries, Ahmedabad, India. Phenobarbitone sodium was purchased from Abbott India Ltd., Mumbai, India. Sodium chloride, potassium chloride, glucose, disodium hydrogen phosphate, potassium dihydrogen phosphate, hydrochloric acid, and glacial acetic acid were obtained from Qualigen Fine Chemicals, Mumbai, India. All the reagents and chemicals used for the study were of analytical grade. Paraformaldehyde, percoll, sucrose, disodium EDTA, Tris buffer, HEPES sodium salt, sodium bicarbonate, calcium chloride, magnesium chloride, 4-aminopyridine, glutamate, NAD⁺ (oxidized form), cresyl violet acetate stain, and luxol fast blue stain were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. Cuprizone, Complete Freund's Adjuvant (CFA), and L-glutamic acid dehydrogenase solution (bovine liver origin) were obtained from Sigma Aldrich, USA. Isoflurane (Raman and Weil Pvt. Ltd., Daman, India) was used for anesthetizing animals. Heparin was purchased from Biological E. Ltd, Hyderabad, India. Deionised water for HPLC was prepared in-house using a Milli-Q integral water purification system (Millipore Elix, Germany).

B16-OVA melanoma cells were kind gift from Dr. Amit Awasthi (TSHTI, India) and were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium (AT162, Himedia, India) with 10% heat-inactivated fetal bovine serum (FBS; RM1112, Himedia, India), 100 U/ml penicillin and 100 μg/ml streptomycin (A002, Himedia, India) and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; MB016, Himedia, India). The LentiX lentiviral packaging cell line were kind gift from Dr. Rahul Purwar (IITB, India) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; AT007, Himedia, India) containing 10% FBS, 2 mmol/L glutamate (TCL030, Himedia, India), 1% non-essential amino acid (ACL006, Himedia, India), 100 U/ml penicillin and 100 μg/ml streptomycin (Himedia, India). Mice splenic T-cells were cultured in complete RPMI 1640 medium (Himedia, India) with 10% heat-inactivated FBS (Himedia, India), 2 mmol/L glutamate (Himedia, India), 1% non-essential amino acid (Himedia, India), 50 U/ml recombinant human interleukin-2 (rhIL-2; 200-02, Peprotech, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Himedia, India).