Computational protocol: In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum

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Protocol publication

[…] Test compound stock solutions were serially diluted in culture medium (twofold: 31.25–1000 μg mL−1). If negative effects on host cell viability were observed or cell proliferation was lower than 75% as determined by the WST-1 assay (Roche Diagnostics GmbH, Vienna, Austria; conducted as described previously []), respective lower concentrations were chosen. All assays were performed in duplicate. When activity of a test substance against C. parvum was found in the initial assays, it was retested twice in independent trials. Monensin sodium salt (purity 90–95%; Sigma-Aldrich, St. Louis, MO, USA) was included in each trial as a positive control (4.2–133.5 nM). For the negative (infected, but untreated) control, unmodified culture medium was used. If solvents or detergents, e.g. ethanol or DMSO, were used to dissolve a test substance, equal amounts were incorporated into controls as well. Test substances were evaluated by a previously published assay [], which is based on an indirect fluorescent antibody technique (IFAT) and the foci detection method [, ]. Briefly, confluent monolayers of HCT-8 cells in 96-well microtitre plates were infected with chlorine-treated C. parvum oocysts before adding the test substance. An inoculum of 2500 oocysts per well was found to produce a maximum number of parasite clusters per area. After incubation for 48 hr (37 °C, 5% CO2, humidified air), the microtitre plates were washed with phosphate-buffered saline (PBS), fixed and incubated with anti-C. parvum antibody from 4b4 mouse hybridoma cells (University of Hohenheim, Germany) [] and FITC-conjugated anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO, USA). Microtitre plates were evaluated under a fluorescence microscope. A cluster of secondary infection (CSI) was defined as a group of five or more green fluorescent dots of about 3–5 μm diameter, which were located in relatively close vicinity. Each well was checked for presence or absence of cluster formation. The lowest concentration of a sample which completely prevented CSI formation was defined as the minimal inhibitory concentration (MIC). For test substances that showed reproducible inhibition of CSI formation, dose-response curves were established (SigmaPlot 6.1, SPSS Inc., Chicago, IL, USA). The percentage of parasite inhibition in wells containing test substances was calculated in relation to negative control wells, which were defined as 100% parasite development. IC50- and IC90-values were calculated by probit regression analysis with 95% fiducial limits (SPSS 10.0, SPSS Inc., Chicago, IL, USA) on the basis of evaluating three microscopic fields per well and counting of the CSIs. Only when IC50- and IC90-values with fiducial limits within the tested concentration range were obtained, was a sample assumed to show significant parasite inhibition. […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Cryptosporidium parvum, Homo sapiens, Olea europaea
Diseases Adenocarcinoma
Chemicals Phenol