Glutamine glutamate glo kit

All studies using animal experiments were reviewed and approved by the University of Houston (UH) IACUC (Institutional Animal Care and Use Committee). We purchased the female 6 to 8-week-old BALB/c mice from Charles River Laboratories. 8 mice were administered PEG-GGT at a dose of 20 mg/kg body weight (b.w). We collected mouse serum at 8, 24, 48, and 72 h. To determine the PEG-GGT concentration in serum, we first prepared a standard curve in which we compared the rate of γGPNA hydrolysis by adding 5 μL of PEG-GGT at different concentrations to 200 μL of 0.5 mM γGPNA in PBS spiked with 5 μL of serum from untreated tumor-bearing mice. We also confirmed that serum from non-treated tumor-bearing mice had no γGPNA hydrolysis activity. To find the concentration of PEG-GGT in the serum of treated mice, we incubated 5 μL of serum in 200 μL of 0.5 mM GPNA in PBS and measured the rate of γGPNA hydrolysis. We determined the concentration of PEG-GGT from the standard curve generated as mentioned above. We determined the circulating glutamine and glutamate levels in serum by enzymatic assay detection kit according to the manufacturer’s protocol (Promega Glutamine/Glutamate Glo kit).

We cultured CT26 cells in 24 well plates to 60% confluency in RPMI-1640 media with 10% FBS. We then washed the cells and replaced the media with RPMI-1640 media without glutamine and 10% dialyzed FBS (dFBS) supplemented with either 2 mM glutamine or 2 mM glutamine and 10 μg/mL PEG-GGT. After 48 h, we washed the cells twice with PBS. We added 200 μL of 80% methanol solution stored at −80°C to the wells and kept the plate in −80°C for 10 min. We then scraped off the cells and vortexed the solution at 4°C for 5 min. We then sonicated (40 kHz) the cells at 4°C for 15 min. Finally, we centrifuged the mixture at 20000 g at 40°C for 10 min. We vacuum evaporated the methanol-water solution and reconstituted the metabolites in 20 μL PBS. We determined the glutamine and glutamate levels in serum by enzymatic assay detection kit according to the manufacturer’s protocol (Promega Glutamine/Glutamate Glo kit). We assayed the total glutathione by an enzymatic assay kit according to manufactures protocol (Cayman total glutathione assay kit: #703002).