Glutamine glutamate glo assay kit

The concentration of intracellular glutamine and glutamate was measured in cell lysates using the glutamine/glutamate-Glo Assay kit (Promega J8021) following producer’s instructions. Luminescence was measured using an iD5 Plate Reader.

Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were
measured using XF Cell Mito Stress Test kit (Seahorse Bioscience) on a Seahorse
XF96 bioenergetic analyzer. MDA-MB-231, HeLa and SiHa cells were pre-incubated
for 24 h in assay medium. Experiments were performed following
manufacturer's instructions. The concentration of glucose and lactate was
measured in cell supernatant using specific enzymatic reactions with a CMA600
analyzer (CMA microdialysis) following producer’s instruction. ATP levels
were measured using the CellTiter-Glo Luminescent Cell Viability assay from
Promega following manufacturer’s instructions on cells that were grown
for 24 h in assay medium containing or not L-glutamine (2 mM),
dimethyl-L-glutamate (2 mM) or dimethyl-2-oxoglutarate (2
mM). All the above metabolic measurements were normalized for total protein
content analyzed at the end of the experiment using the Bio-Rad Protein assay or
the Pierce BCA Protein assay kit (Thermo scientific). The concentration of
intracellular glutamine and glutamate was measured in cell lysate using the
glutamine/glutamate-Glo Assay kit (courtesy of Promega) following
producer’s instruction. Luminescence was measured using a SpectraMax
miniMax 300 imaging cytometer, and data were normalized for cell number.