Nunclon microtiter plates

PBMCs were isolated by density gradient centrifugation on Lympholyte-Mammal medium (1.086 g cm⁻³ at 22 °C, Cedarlane Labs, Skanderborg, Denmark) and cultured in RPMI-1640 with 5 × 10⁻⁵M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES and 10% fetal calf serum (Invitrogen, Taastrup, Denmark), with 2 × 10⁵ cells/well were added in Nunclon microtiter plates (NUNC). The cells were stimulated in triplicates with UV-SvD, Hirep1, CTH93, CT043, CT414_aa605-840 and MOMP in 10 μg ml⁻¹. Synthetic peptides (20-mer) with 10 aa overlap covering CT043, CT414_aa605-840, MOMP and Hirep1 were purchased from (Genscript, Piscataway, NJ, USA) and used for stimulation as pools (5 μg ml⁻¹ of each peptide in the peptide pools) or as individual peptides (5 μg ml⁻¹). The peptides are listed in Supplementary Tables 1. Wells with medium alone and with Staphylococcal enterotoxin B (1 μg ml⁻¹) were included as negative and positive control, respectively. After 3 days of incubation at 37 °C with 5% CO₂, cell culture supernatants were harvested and stored at −20 °C.

Two multidrug resistant non-beta lactamase producing bacterial isolates were subjected to permeability assays. This method was utilized to determine the permeability properties of the outer membrane in the presence of thymol and gallic acid via their increased susceptibility to the bacteriolytic action of detergents [Triton X-100 & sodium dodecyl sulphate (SDS)] and to the cell wall-degrading action of lysozyme. All bacteriolytic agents were purchased from Sigma-Aldrich, St. Louis, USA. The bacteriolytic effect was assayed on Nunclon microtiter plates (Nunc) by measuring the OD630 of bacterial cultures as previously described [21 (link)]. Cell death caused by the sudden influx of these lytic agents was determined by measuring the decrease in optical density (OD) (Relative turbidity %).