Rpmi 1640 cell culture medium

PLGA Resomer RG 503 H, poly(D,L-lactide-co-glycolide), 50:50, Mw 24,000–38,000 was acquired from Evonik, Essen, Germany. SPC 90G (soy phosphatidylcholine, Phospholipon 90G), DDAB (dimethyldioctadecylammonium (bromide salt), and DSPE-PEG 2000 (N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 2000) were purchased from Lipoid, Ludwigshafen, Germany. Ursolic acid was purchased from Wuxi Cima, China. Thiazolyl Blue Tetrazolium Bromide, 4’,6-diamidino −2-phenylindole dihydrochloride (DAPI) and Nile Red were purchased from Merck, Darmstadt, Germany. RPMI-1640 cell culture medium was purchased from Lonza, Verviers, Belgium, Fetal bovine serum, GlutaMAX (L-alanyl-L-glutamine dipeptide in 0.85% NaCl), and 100× antibiotic-antimycotic were purchased from Life Technologies (Gibco/Life Technologies, Warszawa, Poland). Dimethylsulfoxide (DMSO) was purchased from ChemPur, Piekary Slaskie, Poland. Uranyl acetate and copper mesh (400 Mesh) with formware filters and carbon shells were purchased from Agar Scientific, Essex, UK.

Lansoprazole was purchased by Sigma-Aldrich (Milan, Italy) and was dissolved at 20 mM in DMSO in absence of direct light and reconstituted immediately prior its use. Efavirenz was purified from commercially available Sustiva (Bristol-Myers Squibb) as described [3 (link)], dissolved in DMSO and added to the culture medium in the indicated concentrations and times. RPMI-1640 cell culture medium (BE12-702F), antibiotics (DE17-603E), phosphate buffer saline (PBS) (BE17-512F), trypsin/EDTA (BE17-171E) and fetal bovine serum (DE14-701F) were obtained from Lonza (Milan, Italy). Trypan blue was bought from Alexis Biochemicals (Florence, Italy) and Annexin V-FITC Apoptosis detection kit from Enzo Life Sciences (Lausen, Switzerland). 4-Nitrophenyl phosphate disodium salt hexahydrate tablets for proliferation assay was from Sigma-Aldrich.
The pH of all cell culture supernatants were estimated by the use of a pH 123 Microprocessor pH Meter (Hanna Instruments, Italy).

Human metastatic melanoma Me30966 (supplied by Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy) was maintained in RPMI-1640 medium supplemented with 10% fetal calf serum and antibiotics, at 37°C in humidified 5% CO₂. Experiments were performed in buffered (B), unbuffered (UNB) and pH6.5 RPMI-1640 cell culture medium (Lonza). The cell line was negative for mycoplasma contamination, as routinely tested by modified nested polymerase chain reaction (VenorGeM Kit, Minerva Biolabs, Germany).
To allow spheroid formation, 15 × 10³ Me30966 cells were cultured in 96-well plate (Costar Ultra Low Attachment, Sigma.Aldrich) in complete cell culture medium until 72 hours at 37°C and 5% CO₂ in continuous rotation.

HEK293 cells (ATTC CLR-1573) were grown in Dulbecco modified Eagle medium (DMEM) (catalog number 12-604Q; Lonza). The medium was supplemented with a 10% final concentration of fetal bovine serum (FBS) (catalog number 1400-500; Seradigm), 2 mM l-glutamine (catalog number 25-005-Cl; Corning), 100 IU penicillin-streptomycin (catalog number 30-002-CI; Corning), and 100 μg/ml antibiotic–0.25 μg/ml antimycotic (catalog number 30-004-CI; Corning). HK1 cells were grown in RPMI 1640 cell culture medium (catalog number 12-702Q; Lonza) with the corresponding supplements added. The cells were maintained at 37°C with 5% CO₂. HEK293 cells were transfected with plasmids using Lipofectamine 3000 transfection reagent (catalog number L3000015; Invitrogen) according to the manufacturer’s instructions.

AnxA1 was obtained from MyBiosource (San Diego, CA). The compound was stored at -20°C. Prior to use, AnxA1 was immediately diluted in Krebs-Ringer bicarbonate buffer with HEPES (KRB-HEPES, 119 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM HEPES, and 5.5 mM glucose [pH 7.45]) to the required concentration and added to the conjunctival goblet cells. RPMI-1640 cell culture medium, penicillin/streptomycin, trypsin, and L-glutamine were purchased from Lonza (Walkerville, IL). Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). Fura-2 was from Life Technologies (Grand Island, NY). U73122 and U73343, KN92 and KN93 were purchased from Tocris Bioscience (Ellisville, MO). The lectin Ulex Europaeus Agglutinin (UEA-1), histamine, BAPTA/AM, 2-aminoethyl diphenylborinate (2-APB), 1-butanol (1-but), t-butanol (t-but), carbachol, aristolochic acid (AA), thapsigargin and RO317549 were all purchased from Sigma-Aldrich (St Louis, MO). UO126 was obtained from R&D Systems (Minneapolis, MN). H89 and synthetic lipoxin A₄ (LXA₄, 5(S),6(R),15(S)-TriHETE) were from EMD Millipore (Billerica, MA). Resolvin (Rv) D1 (7S, 8R, 17S-trihydroxy-4Z, 9E 11E, 13Z, 19Z-docosahexaenoic acid) was purchased from Cayman Chemical, Ann Arbor, MI). N-Boc-Phe-Leu-Phe-Leu-Phe (BOC2) was obtained from Genscript Corp in Piscataway, NJ.