https://doi.org/10.1038/s41598-022-27090-1
“Gas chromatography-mass spectrometry (GC–MS)
To determine the profile of metabolites, 106 bladder cancer cells before and after cisplatin treatment were trypsinized and counted with a hemocytometer. Then, ice-cold NaCl 0.9% was added to the cells suspended in DMED + 10% FBS with a 5 to 1 volume ratio. This suspension was mixed by gentle inversion and then centrifuged at 1000 g for 1 min. The supernatant was removed and 500 μl ice-cold HPLC grade methanol was added and vortexed vigorously for 30 s. The mix was frozen in liquid nitrogen and rapidly thawed at 37 °C and then centrifuged in 800 g for 1 min at 4 °C. After centrifugation, the supernatant was collected in a cryotube and kept at − 80 °C. The remaining pellet was mixed with ice-cold methanol and supernatant was added to the stored extract at − 80 °C two additional times. Subsequently, a mixture of 500 μl cold HPLC grade chloroform and 250 μl HPLC grade water was added to the pellet remained from previous the step. After freeze–thaw and centrifugation (1000 g, 3 min), the lower phase was gently collected and added to the stored extracts at − 80 °C. The remaining liquid was centrifuged for 1500 g for 1 min at 4 °C and the upper phase was gathered and mixed with previous extracts. This total extract was dried with a mild nitrogen gas flow. For derivatization, the residue was dissolved in 20 μl methoxyamine (Thermo Fisher Scientific, USA) and was heated for 90 min at 30 °C. Next, 50 μl of N, O-bis (trimethylsilyl) tri- fluoroacetamide (BSTFA; Merck, Germany)/ Chlorotrimethylsilane (TMS; Sigma, USA) (49:1 v/v) was added to the samples and were heated at 70 °C for 60 min. The resultants were cooled at room temperature and were applied to the GC–MS instrument (Agilent Technologies, USA). The GC–MS results were analyzed via Mass Hunter software (Agilent Technologies).
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