Table 3.

Recommended experimental design, procedures and minimum reporting standards of a mammalian cell culture metabolomics study, adapted from van der Werf et al. [35], with examples given of each step

Category	Detail	Example
1. Cell culture general growth	Cell line/s used and their source	SH-SY5Y human neuroblastoma, sourced from ECACC
	Configuration of cellular growth	Adherent layer
	Medium and substrates used inclusive of type, supplier, concentrations, additions, supplementations, serum percentage, etc.	DMEM:Ham’s F-12 (1:1) supplemented with 1% v/v 2 mM l-glutamine, 1% v/v 10,000 U/mL penicillin and streptomycin, and 10% v/v FCS, all sourced from Sigma Aldrich
	Growth containers used inclusive of type, supplier, size and geometry	Cells were grown in 75 cm2 tissue culture flasks (sourced from Corning) with 10 mL volume of medium
	Inoculation procedures used, splitting procedures, seeding densities and levels of confluence achieved	For passaging confluent cells and cell counting during experimentation, cells were detached with addition of 1× trypsin/EDTA after first removing the medium and washing the cells with pre-warmed 1× PBS
	Environmental conditions of growth	Cells were kept in a humidified incubator at 37 °C and 5% CO2
2. Characteristics of cells during experimental growth	Growth containers, growth phase of differentiated state	Cells were cultivated in six-well tissue culture plates (sourced from Corning)
	Numbers of culture passage/s and biological replicates	Cell culture passage numbers 28–32 were used for experimentation with four biological replicates per treatment group
	Cell density (optical density or cell number)	Cells seeded at a density of 4 × 105 cells/well in 2 mL of medium
	Depletion of nutrients at treatment time	Plates were left for 24 h to allow for cell adhesion before treatment
	Any phenotypic characteristics specific to the question under study	If carried out, such as differentiation
3. Particulars of the treatment conditions	Nature of the treatment itself e.g. physical stressor, chemical, etc.	Chemical treatment was the neurotoxin insecticide, permethrin
	Dose/s of treatment and vehicle	Permethrin was added as a solution in 100% methanol to a final concentration of 100 µg/mL per well, or 0.25 mM
	Treatment times or intervals	Exposure to permethrin was for 6 and 24 h
	Purity concentration of treatment agent	Permethrin (mixture of cis- and trans-isomers) was purchased at 99.5% purity
	Details of any pre-treatment procedure	Following 24 h post seeding, cells were left for 24 h in 1 mL of serum-free medium before treatment was added
4. Quenching details	The time of sample removal, temperature, and time until metabolic activity ceased	Immediately following exposure time, cell number and viability were determined using the cell number samples, and samples for metabolomics analysis were removed from incubation and cellular activity halted by placing the plates directly onto ice
	Description of quenching technique	Addition of 1 mL 4 °C 1× PBS to each well
5. Sample collection	Discrimination of extracellular from intracellular metabolites	1 mL of medium from each well was collected into one microcentrifuge tube for analysis of extracellular metabolites. Remaining medium removed and adhered cell washed with PBS, before collecting for analysis of intracellular metabolites
	Method of collection and storage	Quenching PBS removed as a wash step, adhered cells were then collected by scraping into 100 µL of additional PBS and transferring into one microcentrifuge tube. All collected samples were immediately snap-frozen and freeze-dried and stored at −80 °C
6. Metabolite extraction procedure	Composition of extraction solution	80% methanol, 20% water solution containing 2.6 µg/mL of 13C6-sorbitol as an internal standard compound
	Description of extraction process	500 µL of extraction solution added to each freeze-dried sample and homogenised in a tissue lyser for 40 s. Extracts were centrifuged and supernatant collected into a fresh microcentrifuge tube. Extracted metabolites were concentrated by evaporating the methanol in a vacuum concentrator
	If present, details of multiple phases collected	Single-phase (methanol/water) extraction was collected
	If known, knowledge of the expected recovery rate and stability of extracted metabolites	If known, should include recovery rate of the metabolites, either targeted for or untargeted, dependant on class of compound, stability in the sample preparation procedure, etc.
7. Biomass normalisation	Details of parallel samples set up next to experimental samples for determination of cell number to use for data normalisation	One extra well per treatment was seeded parallel along with metabolomics samples, for cell counting and viability
8. Sample handling	Any additional clean-up steps undertaken for purification or to protect from degradation	Extracted metabolites were protected from degradation by addition of water and further snap-freezing and freeze-drying
	How the sample are stored when not in use	Once completely dry, metabolite extracts were stored at −80 °C
9. Quality control samples	Details of constituents of QC samples and their use in the analysis	One extra sample from all treatment groups was pooled during extraction procedure and separated into equal volumes for QC samples
10. Expected metabolite detection information	If known, any information on the detection limits, or stability of metabolites expected in the samples	Any expected features in the cell samples (e.g. specific sugars, lipids etc.) that are in relatively high abundance or trace levels, and if it is known if there will be endogenous metabolites that are easily degraded or converted into structurally different features during the analytical process

ECACC European Collection of Cell Cultures, DMEM Dulbecco’s Modified Eagle Medium, FCS foetal calf serum, EDTA ethylenediaminetetraacetic acid, PBS phosphate buffered saline