Agilent qualitative analysis b 07

2D monolayer cultured TIME cells were collected and washed twice with cold PBS and fixed in 80% methanol (Fisher Chemical, LC-MS glade) precooled in dry ice. Cells were detached by scrapping and then transferred to a 2 mL screw cap tube. 3D cultured TIME cells are incubated with Cell Recovery Solution (Corning) for 20 min on ice for depolymerize the Matrigel matrix before fixation. Lysate of cells were quantified by BCA and pelleted by centrifugation (12000 rpm for 10 min at 4°C). The supernatant was transferred into a new 2 mL screw cap tube and driedusing a vacuum concentrator. Dried metabolites were resuspended in acetonitrile/methanol/water(40:40:20).Before applying LC-MS, metabolites were mixed with acetonitrile containing 0.2% folic acid solution and spun down at 12,000 rpm for 10min at 4°C. The supernatant was injected into Agilent Accurate Mass 6230 time of flight (TOF) coupled with Agilent 1290 liquid chromatography (LC) system. Detected ions were deemed metabolites on the basis of unique accurate mass-retention time identifiers for masses exhibiting the expected distribution of accompanying isotopologues. The abundance of metabolites was extracted using Agilent Qualitative Analysis B.07.00 and Profinder B.08.00 software (Agilent Technologies) with a mass tolerance of <0.005 Da.