Glutaminase activity and glutamine measurement assay

Glutaminase activity was determined using Glutaminase (GLS) Assay Kit (Biomedical Research Service). Briefly, two million cells were washed with ice-cold PBS and lysated by 100 μL 1X Cell Lysis buffer on ice for 5 min with gentle agitation. The Supernatant was collected after centrifugation at maximum for 3 min. Followed by measuring the protein concentration, samples were diluted to 0.2-2 mg/ml and 10 μL was used for GLS assay. Samples were combined with 40 μL fresh glutamine solution and incubated at humidified 37°C non-CO₂ incubator for at least 2 h. Followed by adding 50 μL TA Assay solution and incubating for another 2 h, the reaction was stopped by adding 50 μL 3% acetic acid. GLS activity was measured by absorbance at OD₄₉₂ using a plate reader (Versamax).

Tumor glutamine levels were determined using Glutamine Assay Kit (Colorimetric)(Abcam) following the instructions. Briefly, 10-20 mg tumor tissue was washed with cold PBS and resuspended in 10X of ice-cold hydrolysis buffer. Tissue was homogenized and centrifuged for 10 min at 4°C at 10000g. Supernatant was deproteinized by 10kD Spin column. The deproteinized samples will be used for glutamine detection. The absorbance at 450 nm was measured on a microplate reader.