Penicillin streptomycin pen strep

PVA (typically average M_w = 72,000 g mol⁻¹; 98.9% hydrolyzed) was supplied by Biochemica, Germany. Kaolin, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, sodium hydroxide, ethanol, dimethyl sulfoxide (DMSO), and acid citrate dextrose solution (ACD) were purchased from Sigma-Aldrich (Chemie GmbH, Steinheim, Germany). Penicillin–streptomycin (Pen-Strep) (10 KU/10 KU) was obtained from Lonza, Belgium. Yeast extract and tryptone were procured from Bioshop (Canada Inc.). Sodium chloride was provided by Adwic Co. (Egypt).
Gram-negative [Pseudomonas aeruginosa (P. aeruginosa), Shigella sp., and Proteus vulgaris (P. vulgaris)] and Gram-positive [Staphylococcus aureus (S. aureus), and Streptococcus pyogenes (S. pyogenes)] bacteria were used to investigate the antibacterial activities of the formulated materials. The strains were revitalized from glycerol vials by growing overnight at 37 °C and 150 rpm into Luria Bertani (LB) broth medium, consisting of NaCl 10 g l⁻¹, peptone 10 g l⁻¹, and yeast extract 5 g l⁻¹.

RASF were grown out of small biopsies from synovial tissue after joint replacement surgery. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS, Gibco, Waltham, MA, USA), and 100 U/ml penicillin/streptomycin (pen/strep, Lonza, Verviers, Belgium) and used between passage 3 and 8.
Sorted CCR6+ memTh subpopulations were cultured at a density of 2.5 × 10⁴ cells/ml in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS, 2 mM L-glutamine (Lonza), 100 U/ml pen/strep, and 50 μM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Where indicated, the CCR6+ Th memory subpopulations were cocultured with 1.0 × 10⁴ RA synovial fibroblasts (RASF), which were seeded in flat-bottom 96-well plates 24 h earlier. T cells were stimulated with 300 ng/ml soluble anti-CD3 and 400 ng/ml soluble anti-CD28 (Sanquin, Amsterdam, the Netherlands). For some experiments, the cells were cultured with 100 nM 1,25(OH)₂D₃ dissolved in 100% ethanol (Leo Pharmaceutical Products, Ballerup, Denmark), 100 μg/ml secukinumab (anti-IL-17A, Novartis, Basel, Switzerland), or 10 μg/ml anti-IFNγ (R&D Systems, Minneapolis, MN, USA). Controls were an equal volume of 100% ethanol (with a maximum final ethanol concentration of 0,1%) or equal concentrations of corresponding isotype control antibodies, respectively.

Foetal calf serum (FCS) was purchased from Invitrogen Life Technologies, Scotland. Penicillin/streptomycin (Pen/Strep), trypsin/EDTA, minimal essential medium, α-modification (α-MEM) and Medium 199 and other tissue culture reagents were purchased from Lonza, Nottingham, UK. Endothelial cell growth supplement (ECGS) was obtained from Promocell, Heidelberg, Germany. Alkaline phosphatase staining reagents and assay kit and other cell culture reagents were purchased from Sigma Aldrich, Poole, Dorset, UK. Human VEGF-165 was purchased from PeproTech EC Ltd., London, UK. Collagenase B was purchased from Roche Diagnostic Ltd., Lewes, East Sussex, UK. Tissue culture plastics were purchased from Greiner BioOne, okGloucestershire, UK.