Dmem f12 culture medium

OVCAR-3, OAW-42 and SK-OV-3 ovarian cancer cells were obtained from American Type Culture Collection (Manassas, VA, USA). Recombinant huChem-157 was from R&D Systems (Minneapolis, MN, USA), (Cat. No. 2324- CM-025), which is the 16 kDa processed monomeric form of chemerin being most bioactive in the human body (aa 21–157). DMEM/F12 culture medium, FBS, sodium pyruvate, insulin, L-glutamine and Accutase were obtained from Sigma-Aldrich (Munich, Germany). Affinity Script Multi Temperature cDNA Synthesis Kit was from Agilent (Santa Clara, CA, USA). RNeasy Mini Kit, RNase Free DNase Set and Quantitect SYBR Green PCR Kit were obtained from Qiagen (Hilden, Germany). PCR primers were synthesized at Eurofins (Hamburg, Germany).

Main conduit renal and third-order mesenteric arteries were microdissected and cleaned of adherent fat in ice-cold Krebs solution, as previously described.^{24 (link)} Arteries were incubated in DMEM/F-12 culture medium (Sigma Aldrich, Dorset, United Kingdom) supplemented with 1% penicillin-streptomycin (Sigma Aldrich, Dorset, United Kingdom) in a 37°C incubator with 5% CO₂ for 16 hours (for myo-inositol/raffinose treatments) or 48 hours (for gene-silencing experiments). In double knockdown experiments, arteries were incubated in the morpholinos transfection mix for 32 hours and then incubated in medium containing either vehicle or raffinose plus myo-inositol for 16 hours.

Chinese hamster ovary cells (CHO-K1) (ATTC, Manassas, VA, USA) and normal human epidermal keratinocytes (nHEKs) (Cell Systems, Troisdorf, Germany) were used to analyze nucleic acid delivery efficiencies by FLs. Before treatment, CHO-K1 cells were maintained in DMEM-F12 culture medium (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum and a 1/100 dilution of an antibiotic solution (10,000 units penicillin and 10 mg/mL streptomycin in 0.9% NaCl, (Sigma-Aldrich Darmstadt, Germany)). nHEK cells were cultured in DermaLife® K Keratinocyte Culture medium (CellSystems, Troisdorf, Germany) with manufacturer’s supplements lacking tumor necrosis factor (TNF). For experiments on cortical neurons, cells were isolated from rat embryos and cultured as described previously [66 (link)]. All cells were continuously kept at 37 °C and 5% CO2 in a humidified atmosphere. Cell confluence was kept below 80%. Then, 24 h before fusion 60,000 cells were seeded on fibronectin (BD Biosciences, San José, CA, USA) coated Petri dishes (Ø = 3.5 cm) (20 μg/mL fibronectin (BD bioscience) for 30 min at 37 °C) and cultivated.

OptiMEM medium was purchased at Invitrogen (Karlsruhe, Germany). DMEM/F12 culture medium, FBS, sodium pyruvate, insulin, L-glutamine and Accutase were obtained from Sigma-Aldrich (Munich, Germany). DSCAM-AS1 siRNAs were from ThermoFisher (Woodward, PA, USA). Affinity Script Multi Temperature cDNA Synthesis Kit was from Agilent (Santa Clara, CA, USA). RNeasy Mini Kit, RNase Free DNase Set and Quantitect SYBR Green PCR Kit were bought from Qiagen (Hilden, Germany). PCR primers were synthesized at Eurofins Genomics (Ebersberg, Germany). Transfectin reagent was obtained from BioRad (Hercules, CA, USA).

The endometrial tissues from pigs on Day 12 of the estrous cycle were dissected from the myometrium and placed into warm phenol Dulbecco modified Eagle medium/F-12 (DMEM/F-12) culture medium (Sigma, St. Louis, MO) containing penicillin G (100 IU/ml) and streptomycin (0.1 mg/ml) as described previously (5) with some modifications. The endometria were minced with scalpel blades into small pieces (2–3 mm³), and aliquots of 500 mg were placed into T25 flasks with serum-free modified DMEM/F-12 containing 10 μg/ml insulin (Sigma), 10 ng/ml transferrin (Sigma), and 10 ng/ml hydrocortisone (Sigma). Endometrial explants were cultured immediately after mincing in the presence of 0, 2, 20, or 200 nM calcitriol (Enzo Life Sciences, Miami, FL) with E₂ (10 ng/ml; Sigma) and P₄ (30 ng/ml; Sigma) for 24 h with rocking in an atmosphere of 5% carbon dioxide in air at 37°C. Explant tissues were then harvested, and total RNA was extracted for real-time RT-PCR analysis to determine expression levels of endometrial genes. These experiments were conducted using endometria from three gilts, and treatments were performed in triplicate using endometrial tissues obtained from each gilt.

T47D (ATCC^® HTB-133^™), MCF7 (ATCC^® HTB-22^™) and MDA-MB-231 (ATCC^® HTB-26^™) breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). All cancer cell lines were grown in DMEM/F12 culture medium (Sigma, St. Louis, MO, USA) supplemented with 10% FBS (Sigma), penicillin (Sigma) (100 IU/mL), and streptomycin (Sigma) (100 μg/mL) at 37°C in a humidified atmosphere with 5% CO₂.