Cell culture plastics

Experiments were performed using immortalized human umbilical vein endothelial cells HUVECs line EA.hy926 (kindly provided by Dr. CJ Edgell, University of North Carolina, Chapel Hill, NC, USA) [55 (link)]. The cells were routinely maintained in the Earl’s-buffered M199 culture medium, supplemented with amphotericin (2.5 μg/mL), gentamycin (50 μg/mL), L-glutamine (2 mmol/L), hydrocortisone (0.4 μg/mL), and 10% v/v fetal calf serum (Invitrogen, Waltham, MA, USA). The medium was also supplemented with a small dose of ciprofloxacin (0.5 μg/mL) as protection against Mycoplasma intoxication. The cells were cultured in a humid atmosphere with 5% carbon dioxide at 37 °C.
To ensure that BTH, GVH, and RHRextracts were not contaminated with endotoxin, extracts were tested with the LAL assay (Thermo Fisher Scientific, Rockford, IL, USA). The sample was considered as not contaminated when the endotoxin level was less than 0.01 ng/mL. It was considered acceptable and was involved in further in vitro studies.
Unless indicated otherwise, all of the reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture plastics came from Nunc (Roskilde, Denmark) and Costar (Glendale, AZ, USA).

Unless otherwise stated, all chemicals were purchased from Sigma (St. Louis, MO). Cell culture plastics were from Nunc (Roskilde, Denmark). Recombinant human IL-6 and TGF-β1 were from R&D Systems (Abingdon, UK). Neutralizing antibodies as well as appropriate isotype-matched control antibodies were from R&D Systems and Abcam (Cambridge, UK). Bay 87-2243, MG-132 and 3-aminobenzamide (3-AB) were from Bayer Pharma AG (Wuppertal, Germany), Biomol (Plymouth Meeting, PA) and Sigma, respectively.

Human umbilical vein endothelial cell (HUVECs) line EA.hy926 (kindly provided by Dr. CJ Edgell, University of North Carolina, Chapel Hill, NC, USA) was used in in vitro experiments [16 (link)]. The cells were cultured in the Earl’s-buffered M199 culture medium, supplemented with gentamycin (50 μg/mL), amphotericin (2.5 μg/mL), L-glutamine (2 mmol/L), hydrocortisone (0.4 μg/mL) and 10% v/v foetal calf serum (Invitrogen, Waltham, MA, USA). The cells were incubated at 37 °C in a humidified atmosphere of 95 % air and 5% CO2. Cell culture plastics were bought from (Nunc, Roskilde, Denmark) and (Costar, Glendale, AZ, USA), whereas reagents were purchased from (Sigma-Aldrich, St. Louis, MO, USA).