96 well microtitre plate

Murine IL-6 and TNF-α and human IL-6 were quantified using Duoset Kits (R&D Systems) and human IFN-α using an IFN-α pan specific ELISA kit (Mabtech). Murine IFN-α was determined using 96-well microtitre plates (Nunc) coated with monoclonal rat anti-mouse IFN-α (clone RMMA-1, PBL Interferon Source) at 910 ng/ml overnight at room temperature. After blocking with 5% (w/v) BSA/PBS for 1 h at room temperature, 50 μl of supernatant sample was added overnight at 4°C, and detected with polyclonal rabbit anti-mouse IFN-α (PBL Interferon Source) at 80 ng/ml for 2 h, HRP-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories) at 80 ng/ml for 1 h, and BM Blue POD substrate (Roche). Recombinant mouse IFN-α3 (PBL Interferon Source) was included as a standard.

EPEC strains expressing EspA variants were grown overnight in DMEM containing 1 % glucose. Cultures were either mixed together in equal proportions (for initial screening of hybridomas) or used separately (for subsequent testing) and diluted with an equal volume of coating buffer. For testing, 100 µl of mixture was dispensed into each well of 96-well microtitre plates (NUNC Maxisorp), dried overnight at 37 °C, washed twice with PBS and then blocked for 30 min with PBS containing 4 % skimmed milk. Doubling dilutions of serum from test bleeds or undiluted hybridoma supernatants were added to wells and incubated for 1 h, washed three times with PBS, incubated with goat antimouse alkaline phosphatase (Sigma) for 30 min and then washed five times with PBS. Detection was by p-nitrophenyl phosphate (Sigma); reactions were stopped by the addition of 1/4 volume of 3 M sodium hydroxide and absorbance was read at 405 nm.

The cytotoxicity of Q3R against MDCK cells was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [21 (link), 22 ]. The cells were seeded in 96-well microtitre plates (Nunc, Denmark) (3 × 10⁴ cell/well) and incubated at 37 °C in a humidified 5% CO₂ incubator overnight. Then, 2-fold serial dilutions of Q3R in DMEM (100 μl) were added to the cells in triplicate and incubated for more 48 h. Doxorubicin hydrochloride (Pfizer) was used as a positive control. The cells without treatment and cells exposed to dimethylsulfoxide (DMSO) with maximum 0.5% concentration were used as negative and vehicle controls, respectively. After incubation, the colorimetric MTT viability assay was carried out as described before. The cell survival rate was calculated using the following formula: (mean Optical Density (OD) of treated cells/mean OD of control cells) × 100. The 50% cytotoxic concentration (CC₅₀) was defined as the concentration which causes visible morphological changes in 50% of the cells based on the observation under inverted microscope with respect to the control cells. A non-cytotoxic concentration (NCTC) was used for antiviral assays.

AZD6244, BKM120, BYL719, GDC0973, sorafenib, trametinib, BEZ235, Ku-0063794, RAD001, MK2206 and bosentan were obtained from Selleck Chemicals (Houston, TX). Carboplatin and 5-FU were obtained from Sigma. All stock solutions were prepared in DMSO (MP Biomedicals, Solon, OH) at a final concentration in culture media of 0.25% (v/v). Cells in 90 μl medium were seeded (3000 cells/well) onto 96-well microtitre plates (Nunc, Rochester, NY). After 24 hours, 10 μl of medium containing compounds in graded concentrations ranging from 0.1 μM to 1000 μM was added to the wells. Control wells contained 20 μl of relevant solvent to achieve a final concentration of 0.25% of each solvent. The effect on cell numbers was assessed using the CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI) (MTS Assay) at 72 h post-treatment. The IC₅₀ was calculated as the drug concentration that inhibited cell proliferation by 50% compared to vehicle controls as previously described [32 (link)].

Antimicrobial susceptibility testing was performed as minimum inhibitory concentrations (MIC) by micro-broth dilution in 96-well microtitre plates (Trek Diagnostic Systems, Westlake, OH, USA). The following antimicrobials were tested: ampicillin, cefotaxime, ceftazidime, ciprofloxacin, chloramphenicol, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfonamides, tetracycline, and trimethoprim. The results were interpreted according to the European Committee on Antibiotic Susceptibility Testing (EUCAST) epidemiological cut-offs (www.eucast.org) and to Clinical Laboratory Standard Institute [16] or EUCAST clinical breakpoints for those drugs for which epidemiological cut-offs have not been made available (kanamycin, chloramphenicol, sulfamethoxazole, trimethoprim). For streptomycin, a cut-off of 16 mg/L was used, according to EUCAST MIC distributions.
Confirmatory test for the detection of ESBLs were performed on isolates resistant to cefotaxime or ceftazidime according to Clinical Laboratory Standard Institute (CLSI) recommendations [16] .

Antimicrobial susceptibility testing was performed as minimum inhibitory concentrations (MIC) using micro-broth dilutions in 96-well microtitre plates (Trek Diagnostic Systems, Westlake, OH, USA). The following antimicrobials were tested: ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ) ciprofloxacin (CIP), chloramphenicol (CHL), gentamicin (GEN), nalidixic acid (NAL), sulfamethoxazole (SMX), tetracycline (TET), and trimethoprim (TMP). The results were interpreted according to the European Committee on Antibiotic Susceptibility Testing (EUCAST) epidemiological cut-offs (www.eucast.org). EUCAST clinical breakpoints were used for those drugs where epidemiological cut-offs were unavailable: kanamycin, chloramphenicol, sulfamethoxazole, trimethoprim. For streptomycin, a cut-off of 16 mg/L was used, according to EUCAST MIC distributions [18 (link)]. Escherichia coli ATCC 25922 was used was used as a Quality Control strain.
Phenotypic confirmatory tests for the detection of ESBLs were performed on 49 isolates resistant to cefotaxime or ceftazidime according to Clinical Laboratory Standard Institute (CLSI) recommendations [19 ]. Klebsiella pneumoniae ATCC 700603 was used as a Quality Control strain.