At the time of the assay, virus-containing supernatants were thawed and immediately serially diluted in dilution media (unsupplemented Eagle's minimal essential medium for MDCK cells) prior to use in TCID50 assays.
The standard calibration was carried out in horse plasma diluted 1 10 with dilution media, as the instructions of the test kit required dilution of at least 1 10 to minimize plasma matrix interactions with the ELISA.
The anti-diphosphorylated ERK1/2 antibody was diluted in the same dilution media used for light microscopy, but Triton X-100 was omitted from all solutions.
We analyzed each test looking at its endpoint, toxicant and experimental design including replicates, exposure time, number of exposed cysts or organisms and their relative life stage, exposure conditions during hatching and testing (salinity, pH, light intensity, aeration dilution media, and food supply), type of testing chambers, and quality assurance and quality control criteria.
Immediately following the 2-hour incubation period, an additional 100 μl of dilution media was added, and plates were covered with adhesive sealing film (Excel Scientific, Victorville, CA) and returned to 37°C under 5% CO2.
Unimmunized controls were injected with 100 µl PBS i.v. For influenza infections, mice were administered intra-nasally with either A/PR/8/34 (PR8, H1N1 Cambridge) influenza or X31 (H3N2) influenza at the stated dose in 50 µl viral dilution media (VDM; DMEM, 0.1% BSA, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine; all from Sigma, St Louis, MO, USA).
Figure 1 Study of the degradation of the nanoparticle in diluted media after dilution at 2.5 mM.
Working concentrations of 1.5, 3.0 and 4.5 μM LK-A were prepared freshly before each experiment by dilution in media.
Following collagenase treatment, cells were immediately placed on ice and the collagenase activity was stopped by dilution with media containing fetal bovine serum.
All values that were greater than the highest range within the standard curve were repeated after further dilutions with media containing 10% human AB serum.
Confluent MDCK cell monolayers in 96-well plates were washed with PBS and infected with 10-fold virus dilutions (culture media from treated/infected MDCKs or apical washes from treated/infected HAE, see below).
