Rvt5105 refrigerated vapor trap

Mass spectroscopy experiments used human 293 T cells (ATCC) grown in DMEM with 10% FBS. On day zero, 150,000 cells were seeded into 15 cm tissue cultures dishes with 15 mL of either 2.5 µM arsenic or no arsenic media. Each condition had five replicates. On day 3, the no arsenic cells were approaching confluence and required passaging. Arsenic conditions were at ~30% confluence and received a media change. On day seven, both conditions were near confluence, media was removed, and plates were rinsed with ice cold PBS, remaining liquid removed. Plates were frozen at −80°C before processing for mass spectrometric analysis. Cells were scraped off the plates with PBS and pelleted in microfuge tubes. Cell pellets were lyophilized 18–24 hr using a VirTis BenchTop 4K Freeze Dryer and extracted in 100% ethanol using the same sonication program as described for nematode extraction. Following sonication, samples were centrifuged at 20,817 RCF in a refrigerated Eppendorf centrifuge 5417R at 4°C. Clarified supernatant was aliquoted to a new tube and concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting material was suspended in. 1 mL 100% ethanol and analyzed by LC-MS as described. Metabolite measurements can be found in Figure 5—source data 3.

Exo-metabolome (conditioned media) samples were lyophilized ~48 hrs using a VirTis BenchTop 4 K Freeze Dryer. Dried material was directly extracted in 10 mL methanol in 20 mL glass vials stirred overnight. Vials were centrifuged at 2750 × g for 5 min in an Eppendorf 5702 Centrifuge using rotor F-35-30-17. The resulting supernatant was transferred to a clean 20 mL glass vial and concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting powder was suspended in methanol and analyzed directly by HPLC-MS, as described below. Endo-metabolome (nematode bodies) were lyophilized for 18-24 hrs using a VirTis BenchTop 4 K Freeze Dryer. Dried pellets were transferred to 1.5 mL microfuge tubes and disrupted in a Spex 1600 MiniG tissue grinder after the addition of two stainless steel grinding balls to each sample. Microfuge tubes were placed in a Cryoblock (Model 1660) cooled in liquid nitrogen, and samples were disrupted at 1100 RPM for 60 s. This process was repeated two additional rounds for a total of three disruptions. Pellets were transferred to 8 mL glass vials in 5 mL methanol and stirred overnight. Subsequent steps for concentration and resuspension were followed as described for the exo-metablome.