L amino acid quantification kit

The 2.10⁶ RAW264.7 cells were plated on 35-mm plates and mock or MNV infected the next day at an MOI of 10 or washed once and incubated in GCM-dep medium instead of the normal medium for the cells in starvation control. At 10 h p.i., cells were placed on ice, scraped in cold PBS, transferred to a 1.5-ml tube, washed one more times in cold PBS, and the pellets kept at −80°C. The pellets were then resuspended in 300 μl of cold l-amino acid assay buffer according to the manufacturer’s instructions (l-amino acid quantification kit; Sigma), and lysates were then centrifuged at 13,000 rpm for 10 min at 4°C to remove insoluble material. The supernatants were transferred to a new tube, and the amino acids concentrations measured in duplicate on 50 μl of each lysate by adding one volume of the master reaction mix and 30 min incubation at 37°C in the dark. Results were measured at 570 nm in Clariostar plate reader against the kit control standard curve and normalized by the protein contents quantified using the BCA kit (Pierce) according to the manufacturer’s instructions.

Oxaloacetic acid, α-ketoglutaric (2-oxoglutaric) acid, ammonia solution, hydroxyammonium sulfate, amino acids standard solution (Type H), phenyl isothiocyanate (PITC), PTC-amino acid mobile phase solutions A and B, and a Wakosil-PTC column were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Pyruvic and glyoxylic acids were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 4-Metyl-2-oxovaleric acid and an L-amino acid quantification kit were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). An L-glutamate kit YAMASA NEO was purchased from Yamasa Corporation (Choshi, Japan). Electrodes, including gold disks (ø = 3 mm), Pt coils, and Ag|AgCl|sat. KCl, were purchased from BAS Inc., (Tokyo, Japan).