Glutamax suplement

INS-1 cells [obtained from Prof. Claes B. Wollheim] were cultured in Gibco RPMI 1,640 Medium, GlutaMAX Suplement (Thermo Fisher Scientific) with 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich), 1% Penicillin–Streptomycin (Sigma-Aldrich) and 500 μL of 50 mM 2-Mercaptoethanol (Thermo Fisher Scientific). This culture medium contains 11 mM glucose. The culture was maintained at 37 ºC under humidified (5% CO₂, 95% air) conditions. Cell number and viability were measured by an automatic cell counter (EVE, NanoEntek). Cells were subcultured in 175 cm² flasks at a density of 3·10⁶ cells and grown for 7 days with fresh medium supplied every 2–3 days.

Wild type CHO (ATCC, Manassas, Virginia, U.S.) and a serine-SPT deficient CHO cell line, known as LY-B^{8 (link)} (RIKEN BioResource Research Center, Koyadai, Japan), were used. Cell culture products; DMEM:F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) FBS (Fetal Bovine Serum), penicillin, streptomycin, and GlutaMax suplement were purchased from Thermofisher (Waltham, MA). Organic solvents were from Thermofisher (Waltham, MA). All fluorophores; were purchased from Thermofisher (Waltham, MA). Salts for buffer preparation (KCl, NaCl, CaCl_2, HEPES), myriocin, L-α-phosphatidylcholine from chicken egg (PC), sphingomyelin from chicken egg (SM) and D-erythro-sphinganine (sphinganine) were purchased from Sigma-Aldrich (St. Louis, MO, U. S.). All other reagents (salts and Organic solvents) were of analytical grade.