Dulbecco s phosphate buffered saline dpbs

SARS-CoV-2 isolation and expansion were conducted on the Vero E6 cell line, starting from NPS. Briefly, a total of 4 × 10⁵ cells/well were seeded in a 6-well culture multi-well plate and left to adhere overnight in complete medium at 37 °C with 5% CO₂ until 80% confluence was reached. Cell infection was obtained by adsorption of 500 μL of NPS for 120 min at 37 °C with 5% CO₂. After 2 h of adsorption, the inoculum was removed, the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) (EuroClone, Italy), and 2 mL of fresh complete medium was added. Vero E6 cells were maintained for 3 days or more, checking for cytopathic effect (CPE) daily. The procedure was carried out in a biosafety level 3 (BSL3) facility. Infected cell medium was then recovered and stored at −80 °C until use.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine and trypsin/ EDTA were all purchased from Invitrogen, Carlsbad, CA. 200 U/mL penicillin-0.1 mg/mL streptomycin and Dulbecco's phosphate buffered saline (DPBS) were from Euroclone, Milano, Italy. Chemicals used were purchased from Sigma-Aldrich Italy (IT), and were of the highest analytical grade. Standards of chlorogenic acid, quercetin, luteolin, rosmarinic acid, apigenin, and naringenin were purchased from Extrasinthese (Lyon, France). Acetonitrile (Sigma, Milano - Italy) was of HPLC grade. Orthophosphoric acid (ACS ISO, for analysis, 85%) were purchased from Carlo Erba. Water was distilled and filtered through a Milli-Qapparatus (Millipore, Milan, Italy) before use.

Dulbecco’s phosphate-buffered saline (DPBS) (Euroclone, Milano, Italy); Dulbecco’s Modified Eagle’s Medium (DMEM) (LONZA, BioWhittaker^®, Morrisville, NC, USA); Minimum Essential Medium and Non-Essential Amino Acids (MEM, NEAA) (Gibco^®, Life Technologies™. Grand Island, NY, USA); endothelial cell basal medium (EBM) (LONZA, Clonetics^®, Morrisville, NC, USA); fetal bovine serum (FBS) (Life Technologies. Grand Island, NY, USA); L-glutamine, penicillin, streptomycin. Bovine brain extract (BBE), epidermal growth factor (rhEGF), gentamicin sulfate, ascorbic acid, and hydrocortisone (Euroclone, Milano, Italy); (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT), cell proliferation assay ATCC^® 30-1010K kit (Invitrogen Co). Dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA, ).
The human cancer cells HepG2 (hepatocarcinoma cell line) were obtained from ATCC (American Cell Culture Collection), MCF7 (breast cancer cell line) from the pathological anatomy (Civil hospital of Cagliari) and normal human umbilical vein endothelial cell (HUVEC CC-2519) from LONZA (Clonetics^®, Morrisville, NC, USA).