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MB Sample ID: SA255607

Local Sample ID:c1
Subject ID:SU002643
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Cell Strain Details:MDA-MB-231

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Treatment ID:TR002655
Treatment Summary:Cells were placed in static headspace chambers as previously described [4] with new, clean silicon gaskets. Low oxygen, hypoxic gas (1 % O2, 5 % CO2, 94 % N2; purchased from BOC Specialty Gases, Woking, UK) was flushed through the chambers at a rate of 4 L/min for 10 min (chamber volume = 25 L). Chambers were then closed and placed at 37 ˚C for 2 hours to allow residual oxygen in the media to equilibrate with chamber headspace. Chambers were then flushed again at a rate of 4 L/min for 10 min, sealed and returned to 37 ˚C. After a further 24 hours, chambers were flushed again at a rate of 4 L/min for 10 min. 15 ml of gas standards (MeCl, 520 ppb (parts per billion); MeBr, 22 ppb; MeI, 26 ppb; DMS, 110 ppb; CFC-11, 400 ppb and CH3Cl3, 110ppb; BOC Specialty Gases, Woking, UK) were then injected into the chambers through a butyl seal and time zero sample taken. Injected compounds are either known metabolites for cancer cells, or internal standards (CFC-11) for the analysis and quantification of metabolism. Final chamber concentrations were similar to environmental concentrations, e.g MeCl, 1.2 ppb and MeBr 0.05 ppb, particularly more polluted urban spaces (Redeker et al., 2007). Injected gases are the same as those used for calibration. Compounds not injected but detected at first time point, due to residual presence from laboratory air, (including isoprene, acetone, 2-MP, 3-MP and n-hexane) were quantified. Two time zero (T0) samples were taken using an evacuated 500 mL electropolished stainless steel canister (LabCommerce, San Jose, USA) through fine mesh Ascarite® traps (Archbold et al., 2005), after which the chamber was resealed and left on a platform rocker on its slowest setting for 120 min, at which point two further air samples (T1) were collected. Duplicate samples were taken so that two analytical approaches could be performed (targeted and non-untargeted MS). Cells were removed from the chamber, washed with PBS twice and lysed in 500 µL RIPA buffer (NaCl (5 M), 5 mL Tris-HCl (1 M, pH 8.0), 1 mL Nonidet P-40, 5 mL sodium deoxycholate (10 %), 1 mL SDS (10 %)) with protease inhibitor (Sigma-Aldrich, Roche; Mannheim, Germany). Protein concentration of lysates were determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Media alone was treated exactly the same as cells, and only acetone was found to differ significantly between conditions (Supplementary figure 1). These media blank outcomes were subtracted from respective cellular samples prior to protein normalisation. Comparative controls include lab air blanks and those data available from the dataset and collection method published previously which created and quantified metabolic fluxes of volatile compounds from MDA-MB-231 under hyperoxic (lab air) conditions (Issitt et al., 2022a).
Treatment Vehicle:Nitrogen
Cell Storage:37 degrees
Cell Media:DMEM
Cell Envir Cond:Hypoxia/lab air