Coster 3596

Stable hsp105 promoter-luciferase reporter cell lines (pGL105/C3H cells) were cultured in a flat-bottomed 96-well plate (Coster 3596; Corning, NY, USA) and incubated to reach 70 − 80% confluence. The cells were pretreated with test compounds for 30 min and exposed to heat shock at 41℃ for 3 h using a water bath. After washing the cells with PBS (-) (Wako Pure Chemical Industries) twice, 150 µL of 1X Glo Lysis Buffer (Promega, Madison, WI, USA) was added to each well and mixed by shaking for 20 min at room temperature. Luciferase activity and cell viability were measured on the 96-well white plate (136,101; Thermo Fisher Scientific, Waltham, MA, USA) by using a luminometer (GloMax® Discover System; Promega). Luciferase assay reagent (Promega) and CellTiter-Glo® 3D reagent (Promega) were used for the measurement of luminescence and cell viability, respectively.

Biofilms were grown in 96-well polystyrene microtiter plates (Coster 3596, Corning Inc., Corning, NY, USA) following the method described by Sadiq et al [14 ]. Briefly, bacteria were grown overnight in BHI and diluted in the same medium to the OD value of 0.05 using a Multiskan ™ FC Microplate Photometer (Thermo Fisher Scientific). An inoculum volume of 160 μl was used for monospecies biofilms, whereas equal volumes of all strains were mixed to a total volume of 160 μL in case of dual- three- and four-species biofilm combinations as reported (Ren et al., 2015). The microtiter plates were incubated for 24 h at 30 °C and the biofilms were stained with 0.1 % (w/v) crystal violet (CV) and absorbance was measured after solubilising CV with 33 % (v/v) glacial acetic acid at 595 nm (Abs595).