Peptidoglycan pgn

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyp henyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) assay. Briefly, cells were cultured at a density of 6,000 cells/100 µL in 96-well plates pre-coated with PLL and treated with EE and AE at concentrations of 0.5–100 µg/mL, solvent control (0.05% DMSO) and Dulbecco’s phosphate buffered saline buffer. A combination of lipoteichoic acid (LTA) (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) and peptidoglycan (PGN) (Cedarlane Laboratories, Burlington, ON, Canada) (10 µg/mL) was used as a bacterial antigen control and nimesulide (0.5, 1, and 5 µg/mL) was tested as a positive control. After 24 h incubation under 5% CO₂ at 37 °C, the cells were refreshed by adding 100 µL of fresh CGM. Then cells were incubated with 20 µL of MTS reagent (MTS + phenazine methosulfate (PMS)) for 2.5 h. The absorbance was measured at 490 nm and cell viability was calculated using the following equation. ${\displaystyle \text{Cell Viability}\left(\text{\%}\right)=\frac{\text{Absorbance of the treated wells}-\text{Absorbance of the blank}}{\text{Absorbance of the control wells}-\text{Absorbance of the blank}}\times 100,}$ where the treated wells contained cells incubated with test compounds, the control wells contained cells with solvent and CGM, and the blank wells contained CGM only.

Carvacrol, penicillin G sodium salt, DMSO (≥ 99.8%), LTA, ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide, > 95%), poly-l-lysine (PLL), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) and phenazine methosulfate (PMS) were obtained from Sigma-Aldrich Ltd., Oakville, ON, Canada and peptidoglycan (PGN) were from Cedarlane Laboratories, Burlington, ON, Canada. Four S. pyogenes strains ATCC 19615, and ATCC 49399, a clinical isolate (originated from a pharyngitis patient) and an erythromycin-resistant strain (Spy 1558, erm) were used in the study and were grown in brain heart infusion (BHI), Oxoid Ltd., Nepean, ON, Canada. Cultures storage, sub-culturing, and inoculum preparation (1 × 10⁶ CFU/mL) were performed as described previously^{22 (link)}.

Carvacrol (≥98%, food-grade), Dulbecco’s phosphate-buffered saline (DPBS), dimethyl sulfoxide (DMSO), lipoteichoic acid (LTA), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and phenazine methosulfate (PMS) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Peptidoglycan (PGN) was obtained from Cedarlane Laboratories (Burlington, ON, Canada). Human tonsil epithelial cells (HTonEpiCs), tonsil epithelial cell medium, tonsil epithelium cell growth supplement, poly-L-lysine (PLL), trypsin neutralization solution (TNS), and trypsin-ethylenediamine tetra acetic acid (0.25%) solution (TE) were purchased from ScienCell Research Laboratory (San Diego, CA, USA). Fetal bovine serum (FBS) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 7-Aminoactinomycin D (7-AAD) stain was purchased from BioLegend, Inc. (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from different manufacturers: IL-6 and TNF-α ELISA kits were purchased from BD Biosciences (Mississauga, ON, Canada); human ENA-78, GCP-2, and human COX-2 ELISA kits were purchased from RayBiotech, Inc. (Norcross, GA, USA); a human BD-2 ELISA kit was purchased from PromoCell GmbH (Sickingenstraße, Heidelberg, Germany); and PGE₂ and IL-8 kits were purchased from Invitrogen (Nepean, ON, Canada).