Glutamine and glutamate determination kit

Intrasynaptosomal glutamate and glutamine content was measured with Glutamine and Glutamate Determination Kit according to manufacturer’s instructions (Sigma-Aldrich, Germany).
Glutamate release from synaptosomal preparations was measured by fluorometric assay essentially as described by Nicholls et al. (1987 (link)). Briefly, ~100 μl synaptosomes (~1 mg/ml) suspended in aCSF supplemented with 2 mM NADP⁺ and 6.32 U L-glutamic acid dehydrogenase (and 2 mM CaCl₂ whenever appropriate) was distributed into each of the 96 wells. Synaptosomes were depolarized with 30 mM KCl 5 min thereafter and the increase in NADPH fluorescence was monitored over a 10 min time period. Fluorescence emission was recorded at 450 nm and the excitation wavelength was set at 360 nm. Each experimental condition was carried out in triplicate on the same plate using Victor X3 fluorometer (Perkin Elmer, USA). The amount of released glutamate was calculated based on calibration curves prepared in parallel and the enzyme lag (Nicholls et al., 1987 (link)) was accounted for when converting rates to nmol/mg protein/10 min. The results obtained in the presence of 2 mM CaCl₂ reflect total glutamate release whereas in the absence of Ca²⁺ in the reaction mixture as Ca²⁺-independent glutamate release.

Glutamine and Glutamate Determination Kit and α-ketoglutarate (α-KG) Assay Kit were bought from Sigma-Aldrich. According to the manufacturer’s instruction, the levels of glutamine, glutamate, and α-KG were determined.

Glutamine metabolism was assessed by measuring glutamine intake and glutaminase activity using the Glutamine and Glutamate Determination Kit (Sigma-Aldrich, Shanghai, China) and the PicoProbeTM Glutaminase (GLS) Activity Assay Kit (BioVision, Milpitas, CA, USA) were evaluated by measuring glutamine intake and glutaminase activity according to the manufacturer's instructions. Results were normalized by the number of cells in each reaction. Experiments were performed in triplicate and repeated three times.

A glutamine and glutamate determination kit was purchased from Sigma (GLN1). An ATP determination kit was purchased from Thermo Fisher (AZZ066). The lactic acid assay kit was purchased from the Nanjing Jiancheng Bioengineering Institute (A019-2). All assays were performed following protocols described in the respective instruction manuals. NMR analysis for the concentrations of different metabolites was conducted in Wuhan Anachro Technologies INC.

Cells were plated in 6-well plates at 4 × 10⁵ cells per well, and the medium was changed to complete medium (2 mM Gln) on the following day. The cell number was counted, and medium was collected and analyzed using the Glutamine and Glutamate Determination Kit (GLN1, Sigma) after 24 hours. Gln consumption was compared with that in medium without cells under the same condition and normalized to the cell number.

For the quantification of glutamine, macrophages (5 × 10⁵/well in 24-well plates) were infected for 15 min, 30 min, 1h, 2h, 6h, and 24h at 37°C for 5% CO₂ with live A. fumigatus conidia at a 1:10 effector-to-target ratio. After infection, supernatants were collected and centrifuged, and glutamine levels were quantified using the Glutamine and Glutamate Determination kit (Sigma-Aldrich), following the manufacturer’s instructions.