Hyamine

An aliquot of 50 μl of the respective P. luminescens overnight culture diluted to an OD₆₀₀ of 1.0 was spread in a Z pattern onto the surface of a lipid agar plate [1% (v/v) corn syrup; 0.5% (w/v) yeast extract; 5% (v/v) cod liver; 2% (w/v) MgCl₂ × 6 H₂O; 2.5% (w/v) Difco nutrient agar (Becton Dickinson, Heidelberg)] using an inoculating loop. The plates were incubated at 30 °C for 3 days before adding 50 surface sterilized infective juvenile nematodes (IJs) to the bacterial biomass. Nematodes were surface-sterilized by washing in a solution [0.4% (w/v)] of hyamine (Sigma-Aldrich, Deisenhofen)]. The plates were kept at room temperature. Nematode recovery was assessed 7–8 days after addition of IJs by counting the number of hermaphrodites on the lipid agar plate.

Compound cytotoxicity was assessed in a BSL-2 counter screen as follows. Host cells in media were added in 25 μL aliquots (4,000 cells/well) to each well of assay ready plates prepared with test compounds as described above. Cells only (100% viability) and cells treated with hyamine (Sigma cat# 1622) at 100 μM final concentration (0% viability) served as the high and low signal controls, respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant concentration for all wells as dictated by the dilution factor of stock test compound concentrations. After incubating plates at 37°C/5% CO2 and 90% humidity for 72 hours, 30 μL Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a BMG PHERAstar plate reader following incubation at room temperature for 10 minutes to measure cell viability.