Heraeus megafuge 1

The human colorectal carcinoma cell line (HCT116) was purchased from the American Type Culture Collection (ATCC, USA) and routinely cultured in RPMI-1640 medium (Invitrogen, USA) containing 10% (v/v) foetal calf serum (FCS; Thermo, USA). When 80% confluence was reached, cells were detached by incubation with 5 mM trypsin/EDTA and harvested after centrifugation in a Heraeus Megafuge 1.0 (Thermo Scientific, USA) at 1200 rpm for 5 min at room temperature. Cells were resuspended in media and viable cells counted using a haemocytometer and trypan blue staining. All cell lines were confirmed negative for mycoplasma contamination.

A 1-mL volume of the e-liquids bought during the monitoring of the online market was extracted by adding 1 mL of methanol and 1 mL n-hexane using an overhead shaker for 5 min at lowest rotation speed (Reax 2; Heidolph, Schwabach, Germany). Subsequently, samples were centrifuged at 2860 × g for 5 min (Heraeus Megafuge 1.0; Thermo Scientific, Schwerte, Germany). Ten microliters of the supernatant were evaporated to dryness at 40 °C under a gentle stream of nitrogen. Prior to analysis by the GC–MS system (injection volume 1 µL), the samples were reconstituted in 100 µL of dry ethyl acetate.
For NMR analysis, 1 mL of an e-liquid was extracted five times using n-hexane/ethyl acetate (99:1, v/v). The combined supernatants were evaporated to dryness and resulted in 4 mg of a raw extract containing 5F-Cumyl-PINACA, which was used for structure elucidation by NMR spectroscopy.

The mouse motor neuron-like cell line (NSC-34), a hybrid cell line produced by fusion of neuroblastoma with mouse motor neuron-enriched primary spinal cord cell (Cashman et al., 1992 (link)), was routinely cultured in DMEM/F12 supplemented with 10% (v/v) FBS. The human embryonic kidney (HEK293) cell line, which has been used extensively as an expression tool to study recombinant proteins, was cultured in the same media. Cells were maintained in an incubator at 37°C under a humidified atmosphere containing 5% (v/v) CO₂. When 80% confluence was reached, cells were detached by incubation with 5 mM Trypsin-EDTA and harvested after centrifugation in a Heraeus Megafuge 1.0 (Thermo Scientific, USA) at 1,200 rpm for 5 min at RT. Cells were resuspended in media, and viable cells counted using a hemocytometer and trypan blue staining. Cells were confirmed free of Mycoplasma contamination.