0.1 μm spin filter

A modified Bligh and Dyer protocol24 (link) was used for the extraction of lipid mediators from VC. Briefly 10 μl of internal standard mix for all 3 analytical platforms were added to 15 mg of VC and dissolved in 0.19 mL of CHCl₃. Samples were then vortexed for 30 sec and sonicated in an ultrasound bath for 10 min. Afterwards 0.38/0.15/0.19/0.19 mL of MeOH/H₂O/CHCl₃/H₂O were sequentially added with 30 sec vortex for each step. Samples were centrifuged at 3000 rcf for 5 min and the organic phase was withdrawn. VC was re-extracted with 0.30 mL of CHCl₃ and organic extracts were combined, concentrated under vacuum, resuspended in 100 μl of MeOH and filtered using 0.1 μm spin filters (Merck Millipore, Billerica, MA, USA) before analyzing via the lipid mediator platforms described below. Six vernix quality controls (QC) were pooled from two children not included in the study and used to control for quantification reproducibility and batch extraction effects.

For all experiments, we thawed protein aliquots on ice freezer stocks and then either dialyzed in the appropriate experimental buffer overnight at 4 °C or exchanged 3× into experimental buffer using 3K microsep spin concentrator columns (Pall Corporation). We filter sterilized all samples using 0.1-μm spin filters (EMD Millipore). Thawed aliquots were used for no more than 1 wk before they were discarded. We measured concentration by placing the protein into 6 M guanidinium HCl and measuring absorbance at 280 nm. We used an extinction coefficient of 6,990 M⁻¹ cm⁻¹, calculated using the ExPASy ProtParam tool.