Lecithin

Animals were cultured under non-starved conditions for at least 4 generations before cold and freezing resilience assays. For cold resilience assay, bleach-synchronized L4 populations were kept at 4 °C for 20 h and then recovered for 24 h at 25 °C. For freezing resilience assay, bleach-synchronized L4 populations were kept at −20 °C for 45 min and then recovered for 24 h at 25 °C. For both cold and freezing experiments, NGM plates spread with equal agar thickness seeded with equal amounts of OP50 were used while cold and freezing temperature readings were monitored by thermometers to ensure minimal fluctuation. After cold or freezing shock, animals were moved to 25 °C for recovery and scored as dead if they showed no pumping and movement upon light touch with the body necrosis subsequently confirmed by light microscopy. For phospholipid and Lecithin rescue experiments, phospholipid (11145, Sigma-Aldrich), Lecithin (O3376-250, Fisher Chemical) or PC (P5394-10G, Sigma-Aldrich) was prepared as mixture by dissolving in M9 solution (from 1 to 20 mg/ml) and thorough vortexing. Phospholipids or Lecithin mixtures were then added (200 µl/60 cm plate) on NGM plates with pre-seeded OP50 and dried briefly before placing animals for cold or freezing tolerance assays.

Hydrogenated palm oil (Softisan 154) was obtained from Condea (Witten, Germany). Olive oil (Basso) was obtained from Basso Fegele and Figli Srl (San Michele Di Serino, Italy). Eagle's minimal essential medium (EMEM), thymoquinone (TQ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder, trypan blue dye solution, propidium iodide (PI), thimerosal, and sorbitol were purchased from Sigma-Aldrich (St. Louis, USA). RPMI-1640 tissue culture medium, penicillin/streptomycin antibiotic, Mycoplex foetal bovine serum (FBS), and trypsin-EDTA were purchased from PAA Laboratories (Linz, Austria). Other reagents used were lecithin, a form of phospholipid (Cologne, Germany), nonionic surfactant Polysorbate 80 (Fisher-Scientific, USA), and HPLC grade methanol (Merck, USA).

AgNO₃, chlorhexidine digluconate, 20% (w/v), HNO₃, ~70% (w/w) were purchased from Sigma-Aldrich. Chemicals for the preparation of the neutralization solution: sodium thioglycolate, sodium thiosulfate, lecithin, were purchased from Acros Organics; Tween 80 (polyethylene glycol sorbitan monooleate) was purchased from Fluka.