Thiazolyl blue mtt

THP-1 monocytes were cultured in 10% heat-inactivated FBS RPMI-1640 medium supplemented with penicillin-G 100 units/ml and streptomycin 100 μg/ml. HUVEC were cultured in (1%) low serum EndoGro™ medium, culture kit purchased from Merck Millipore™ (Paris, France). Micro-BCA Protein Assay Kit was purchased from Fisher Scientific™ (Illkirch-Graffenstaden, France). TNF-α was purchased from PeproTech™ (Neuilly-sur-Seine, France). Non-conjugated guinea pig anti-MMP-9 EP1255Y mAb was purchased from antibodies-online (Aachen, Germany). The cy3-conjugated human anti-CD31/PECAM-1 (platelet endothelial cell adhesion molecule-1) mAb (clone 9G11), and streptavidin-fluorescein were purchased from bio-Techne™ (Abingdon, United Kingdom). SB-3CT (commercial MMP-2, -9 inhibitor) was purchased from Enzo Life Sciences™ (Villeurbanne, France). RES was purified from stalks of Vitis vinifera, Vitaceae, according to the process described by Delaunay and colleagues [26 (link)]. JNK inhibitor (sp600125), DMSO, MTT (Thiazolyl Blue), all chemicals and solvents used during the synthesis individual NADES components were purchased from Sigma–Aldrich™ (Marnes la Coquette, France). All other chemicals used in the present study are of molecular biology grade.

Human polymorphonuclear (PMN) cells from fresh venous blood of healthy adult volunteers were isolated according to Drewniak et al. (78 (link)), with modifications. Cells were harvested by centrifugation in isotonic Percoll, lysed, and resuspended in HEPES-buffered saline solution. A. nidulans asexual spores were incubated with PMN cells (1 × 10⁵cells/ml; ratio of 500 PMN:1 conidium) in a 96-well plate overnight at 37°C in RPMI 1640 medium containing glutamine and 10% fetal calf serum (Life). PMN cells were lysed in a solution of water and sodium hydroxide (pH 11.0) (Sigma-Aldrich), and spore germination was determined using an MTT (thiazolyl blue; Sigma-Aldrich) assay. Strain viability was calculated relative to incubation without PMN cells, the level of which was set at 100% for each sample. The viability of A. nidulans germinated spores in the presence of PMN cells was determined as described previously (32 (link)).

The cytotoxicity of the isolates was tested using MTT assay reported by Mossmann [23 (link)] against the immortalized human skin epithelial keratinocytes (HaCaT) cells. The HaCaT cells were cultured in Dulbecco’s modified Eagle medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution. The HaCaT cells were grown for 4–5 days at 37 °C in a 5% carbon dioxide atmosphere. The cells were removed from the culture using the trypsin–ethylenediaminetetraacetic acid solution and transferred into a 96-well plate. The tested samples were applied to the cells and incubated at 37 °C for 24 h. Thiazolyl blue (MTT, SIGMA), dissolved in phosphate-buffered saline solution (PBS) at a final concentration of 0.8 mg/mL, was added to the cells that were exposed to the isolated compounds before incubation at 37 °C in the dark for 4 h. After observing the MTT change in color (blue to purple), the media were separated and washed with PBS. The produced formazan salts were dissolved with DMSO, and their concentrations were obtained by measuring their absorbance at 570 nm in a spectrophotometer. The IC₅₀ values of the tested compounds and the positive control tamoxifen were determined using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA).

LPS and thiazolyl blue (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum and penicillin/streptomycin solution were from Hyclone. All molecular reagents were obtained from Toyobo (Osaka, Japan) or TAKARA (Otsu, Japan). Fruits were purchased from a local market.