Rpmi with l glutamine

Sphingosine 5/6-fluorescein (SF) and sphingosine-1-phosphate 5/6-fluorescein (S1PF) were procured from Echelon Biosciences Inc. (Salt Lake City, UT). Monosodium phosphate, 1-propanol, isopropanol, methanol, and pre-cleaned glass slides (50 mm × 45 mm × 1.5 mm) were acquired from Fisher Scientific (Pittsburgh, PA). Sodium chloride, sodium azide, potassium chloride, paraformaldehyde, magnesium chloride, calcium chloride, piperazine-1-ethanesulfonic acid (HEPES), formic acid, and glucose were purchased from Sigma-Aldrich Inc. (St Louis, MO). Ethanol was procured from Decon Labs (King of Prussia, PA). Roswell Park Memorial Institute Media (RPMI) with l-glutamine, fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline, and penicillin/streptomycin were purchased from Invitrogen (Grand Island, NY). EPON resin 1002F (phenol, 4,4′-(1-methylethylidene)bis-, polymer with 2,2′-[(1-methylethylidene)-bis(4,1-phenyleneoxymethylene)]bis-[oxirane]) was acquired from Miller–Stephenson (Sylmar, CA, USA). SU-8 developer (1-methoxy-2-propyl acetate) was purchased from MicroChem (Newton, MA, USA). Poly(dimethylsiloxane) (PDMS) (Sylgard 184 Silicone Elastomer) was procured from Dow Corning (Midland, MI). Acetonitrile was purchased from VWR (Radnor, PA).

HEK293T, HeLa, NIH-3T3, (ATCC) were cultured in DMEM containing glucose and L-glutamine (Corning) supplemented with 10% FBS, 1% penicillin and streptomycin. K562 (ATCC) cells were cultured in RPMI with L-glutamine (Life Technologies) supplemented with 10% FBS, 1% penicillin and streptomycin. Primary cortical neurons (PCN) were isolated as previously described³⁸ and maintained in Neural Basal media supplemented with 1× B27 supplements, 1% penicillin and streptomycin and 1% Glutamax (Thermo Fisher Scientific). All cell lines were grown at 37 °C, in an atmosphere of 5% CO₂.