Green cmfda

The leukocyte–endothelial cell interaction was investigated using a leukocyte cell adhesion assay. In brief, confluent HUVECs were pretreated with S. pilosa extracts for 30 min followed by the inflammatory activation of HUVECs by TNF (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) for 24 h. THP-1 cells were fluorescence-labeled using Green CMFDA (Cayman Chemical, Ann Arbour, MI, USA). Then, 1.5 × 10⁵ THP-1 cells per well were allowed to adhere to the HUVEC monolayer for 5 min. Non-adherent THP-1 cells were washed away and the relative amount of adhered THP-1 cells was determined by fluorescence measurement (ex: 485 nm; em: 535 nm) using a microplate reader (SPECTRAFluor Plus, Tecan, Männedorf, Switzerland).

Dimethyl sulfoxide (DMSO), absolute ethanol, glacial acetic acid, propidium iodide, staurosporine and triton X-100 were of analytical grade and were purchased from Sigma-Aldrich (Merck KGaA, USA). Neutral red dye was from Apollo Scientific Sigma (Bredbury, Stockport, Cheshire, UK). Sodium citrate was obtained from Carl Roth (Karlsruhe, Germany); collagenase A was from Roche (Basel, Switzerland). Τhe Collagen G used for pre-coating the plastic ware on which HUVECs were grown was from Biochrom (Berlin, Germany). Recombinant human TNFα was purchased from PeproTech (Rocky Hill, NJ, USA) and the fluorescent dye Green CMFDA was obtained from Cayman Chemical (Ann Arbour, MI, USA).