96 well microtiter plate

Intracellular TG2 transamidase activity was measured as previously described [34 (link)]. Briefly, the cells were seeded in Greiner 96-well microtiter plates (Greiner Bio-One, Kremsmuenter, Austria) and incubated with 0.2 mM 5BAPA and 0.1 mM aminoguanidine overnight at 37 °C. The cells were treated with increasing concentrations of ACR in serum-free media for 4 h and then fixed with a 10% formaldehyde solution, permeabilized, blocked, and stained with streptavidin-tetramethylrhodamine isothiocyanate (TRITC, Jackson ImmunoResearch Laboratories). Intracellular TG2 transamidase activity was then detected as a fluorescence signal from TRITC and analyzed using an ImageXpressMICRO High Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using a Multi-Wavelength Cell Scoring Application Module in MetaXpress Image Analysis software (Molecular Devices).

The cells were seeded in Greiner 96-well microtiter plates (Greiner Bio-One) and treated with ACR for 4 h. Then, the cells were loaded with a calcium-sensitive fluorescent ratiometric Fura-2AM dye (Dojindo Molecular Technologies) for 30 min at 37 °C. The fluorescence intensities were determined by recording at 340- and 380-nm excitation and 510-nm emission with the EnSight Multimode Plate Reader (Perkin Elmer Inc.). The ratio of F340 to F380 (F340/F380) was used as a relative indicator of intracellular Ca²⁺ concentration.

This study was carried out in accordance with the guidelines of the National Health and Medical Research Council (NHMRC) of Australia. The protocol was approved by Monash University’s Psychology Animal Ethics Committee. Primary astrocyte cell cultures were derived from newborn Wistar rat pups obtained from Monash Animal Services. Constituents of growth media were obtained from Gibco (Carlsbad, CA, USA): Dulbecco’s modified Eagle medium (DMEM), fetal calf serum (FCS), streptomycin sulfate and penicillin G. Triton X-100 was obtained from Ajax Finechem (Seven Hills, Australia). Lead (II) acetate trihydrate, BSO, 3AT, H₂O₂ and all other chemicals were obtained from Sigma (Australia). Twenty four-well cell culture dishes and 96-well microtiter plates were obtained from Greiner Bio-One (Frickenhausen, Germany).

Pre- and post-vaccination (first dose) plasma rotavirus-specific IgA titers were determined by ELISA using a modification of the method described by Bernstein et al.^{45 (link),46} . Briefly, 96-well microtiter plates (Greiner Bio-One, Kremsmünster, Austria) were coated with guinea pig anti-rotavirus antibody (SBL, Stockholm, Sweden). After blocking, either RV1 or RV5 (1:100) was added and incubated at 37 °C for 1 h. Serially double diluted plasma (100 µl; 1:20–1:640 for RV1 or 1:50–1:1,600 for RV5) was added and incubated at 37 °C for 1 h. Horseradish peroxidase-conjugated goat anti-human IgA (P0216; Dako, Glostrup, Denmark) and 1-Step™ Ultra TMB-ELISA Substrate (Thermo Fisher Scientific, Stockholm, Sweden) were used as the detection system. The titer was defined as the reciprocal of the highest plasma dilution having OD₄₅₀ ≥ 0.100. Plasma IgA titers of ≥80 or ≥100 in the RV1 or RV5 cohorts, respectively, were defined as rotavirus-specific IgA seropositive and a 4-fold increase from pre- to post-vaccination was defined as seroconversion. PBS was included for background monitoring. Breast-milk rotavirus-specific IgA titers were determined following the same procedure.

Single colonies of the S. cerevisiaehis6Δ strain (used here as the appropriate wild type [WT] background) and of deletants of interest, all isogenic with S. cerevisiae BY4743 (MATa/αhis3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0), were used to inoculate YPD broth cultures (pH 4.0) in Erlenmeyer flasks and incubated overnight at 30°C with orbital shaking at 120 rpm. Overnight cultures were diluted to an OD₆₀₀ of 0.5 and cultured for a further 4 h in fresh medium. These 4-h mid/late-exponential cultures were diluted to an OD₆₀₀ of 0.2, and 75-μL aliquots were transferred to 96-well microtiter plates (Greiner Bio-One, Stonehouse, United Kingdom) that had already received aliquots of 75 μL medium containing the antimicrobial dissolved in DMSO, such that 2% of the final 150-μL volume was DMSO, to give final antimicrobial concentrations as specified in Results. Plates were incubated at 30°C with shaking in a BioTek Powerwave XS microplate spectrophotometer, and the OD₆₀₀ was recorded every 30 min. Growth assays of deletants were performed in three biological replicates (each performed in three technical replicates). The significance of the phenotypes was verified using two-tailed t tests where an equal variance was assumed (P < 0.05).

Clinical bacterial isolates were used in a MIC assay to investigate the sensitivity of bacterial pathogens from the equine respiratory tract to eBDs. The test organisms included the Gram-positive bacteria Streptococcus equi subsp. zooepidemicus (strain 4001), Streptococcus equi subsp. equi (strain 3830), Staphylococcus aureus (strain 3939), and Rhodococcus equi (strain 3851) and the Gram-negative bacteria Actinobacillus equuli subsp. equuli (strain 4005) and Bordetella bronchiseptica (strain 3033). All organisms were grown on Oxoid sheep blood agar plus plates (Thermo Fisher Scientific) overnight in a humidified incubator (5% CO₂) at 37°C. Inocula were prepared by swabbing bacterial colonies from the plates, suspending them in PBS (turbidity equivalent to a 0.5 McFarland standard), and diluting this suspension 1:100 in Mueller-Hinton II (MH) broth (BD Biosciences, San Jose, CA, USA), achieving a final concentration of approximately 10⁶ CFU/ml. eBDs were serially 2-fold diluted in MH broth at concentrations ranging from 200 μg/ml to 0.02 μg/ml and pipetted into 96-well microtiter plates (Greiner Bio, Kremsmünster, Austria). The inoculum was added to the eBD dilutions 1:1, and the bacteria were incubated in a humidified incubator (5% CO₂) at 37°C for 24 h. MIC values were determined as the lowest concentration of eBD at which there was no visible bacterial growth.