Minimum essential medium eagle

The promyelocytic leukemia HL-60 and breast cancer adenocarcinoma MCF-7 cell lines were purchased from the European Collection of Cell Cultures (ECACC). Leukemia cells were cultured in RPMI 1640 plus GlutaMax I medium (Gibco/Life Technologies, Carlsbad, CA, USA). MCF-7 cells were maintained in Minimum Essential Medium Eagle (Sigma Aldrich, St. Louis, MO, USA) and supplemented with 2 mM glutamine and men non-essential amino acid solution (Sigma Aldrich, St. Louis, MO, USA). Both media were supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Beit-Haemek, Israel) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Sigma-Aldrich, St. Louis, MO, USA). Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC). Cells were cultured using EGM-2 Endothelial Medium BulletKit purchased from Lonza (Lonza, Walkersville, MD, USA). Cells were maintained at 37 °C in 5% CO₂ atmosphere and grown until 80% confluent.
The tested compounds were dissolved in sterile dimethyl sulfoxide (DMSO) and further diluted with culture medium. The final concentration of DMSO in cell cultures was less than 0.1% v/v. Controls without and with 0.1% DMSO were performed in each experiment. At the used concentration, DMSO had no effect on the observed parameters.

A-498 cells (ATCC, HTB-44) were cultured in Minimum Essential Medium Eagle (Sigma-Aldrich) plus 10% fetal bovine serum (FBS, ThermoFisher Scientific) and 100 U/mL penicillin/streptomycin (ThermoFisher Scientific). 786–0 (ATCC, CRL-1932), NCI-H1299 (ATCC, CRL-5803), CT26 (ATCC, CRL-2638) and 4 T1 (ATCC, CRL-2539) cells were cultured in RPMI Medium 1640 (ThermoFisher Scientific) plus 10% FBS, 1 mM sodium pyruvate (ThermoFisher Scientific), 0.45% D-(+)-Glucose (Sigma-Aldrich) and 100 U/mL penicillin/streptomycin. HEK293T (ATCC, CRL-3216) and LL/2 (ATCC, CRL-1642) cells were cultured in Dulbecco’s Modified Eagle Medium (ThermoFisher Scientific) plus 10% FBS and 100 U/mL penicillin/streptomycin.

Cortical neurons were obtained from newborn rats (Sprague-Dawley) within 24 hours after birth using mechanical and enzymatic procedures described in earlier studies^{60 (link)}. Rats were anesthetized by CO₂ inhalation according to protocols approved by the Technion’s ethics committee. All procedures involving cell preparation and animals handling were performed in accordance with these guidelines and regulations. The neurons were isolated and plated directly onto substrate-integrated multi electrode arrays. They were allowed to develop into functionally and structurally mature networks over a period of 2 weeks and were used in experiments within the period of 2–6 weeks post plating. The plated neurons cover an area of about 380 mm², bathed in Minimum Essential Medium Eagle (Sigma), supplemented with heat-inactivated horse serum (5%), glutamine (0.5 mM), glucose (20 mM), and gentamycin (10 μg/ml), and maintained in an atmosphere of 37 °C, 5% CO₂ and 95% air in an incubator as well as during the recording phases. An array of 60 Ti/Au extracellular electrodes, 30 μm in diameter, spaced 500 μm from each other (MultiChannelSystems, Reutlingen, Germany) was used. The insulation layer (silicon nitride) is pretreated with polyethyleneimine (Sigma, 0.01% in 0.1 M Borate buffer solution).

Primary cultures of human rectal smooth cells (HRSMC) were obtained from ScienCell^TM (Catalogue Number: 2980). Cells were cultured in either proliferation medium consisting of Minimum Essential Medium Eagle (Sigma Aldrich, UK) supplemented with 10% foetal bovine serum (FBS) (Life Technologies Ltd, UK), 1× non‐essential amino acids (NEAA), 2 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin (Sigma Aldrich, UK), or differentiation medium consisting of Dulbecco's Modified Eagle Medium (Sigma Aldrich, UK) supplemented with 1× NEAA, 2 mM glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, and 2 ng/mL transforming growth factor (TGF)‐β (PeproTech EC Ltd, UK).

Caco-2 cells were cultured to a tight monolayer on the upper transwell filter of a Millicell-24 cell culture plate (Merck-Millipore, Darmstadt, Germany) and maintained for approximately 14 days until stable transepithelial electrical resistance (TEER) was achieved. The standard culture medium (Minimum Essential Medium Eagle, Sigma, supplemented with 10% FBS (Biosera, Kansas City, MO, USA), 1% penicillin/streptomycin (Gibco), and 1% MEM-NEAA (Gibco) was used and changed every 2 days. An epithelial volt-ohm meter (Millicell^® ERS-2, Merck-Millipore) was used to measure the TEER of the cell monolayer. The cells with stable TEER were then exposed to 300 μM of NSAIDS, where 0.3% DMSO was a diluent control.