Multiskan ex microplate photometer

MIC values were established using a macro broth dilution method in cation-adjusted Müller–Hinton (MH) broth. 96-well plates (200 μL of 128 μg mL^–1, 64 μM) complex in MH broth, diluted 2ⁿ μg mL^–1, inoculated with each bacterial strain (10³ cfu mL^–1) were sealed and growth was monitored over 20 h at 37 °C with an iEMS 96-well plate reader (see ESI†).
IC₅₀ values were determined by incubating cells in 96-well plates (2.0 × 10³ cells per well) for 24 h at 37 °C, 5% CO₂ prior to drug exposure. Compounds were added (100 μM to 5 nM in cell medium) for a further 96 h. 3-(4,5-Dimethylthiazol-1-yl)-2,5-diphenyl tetrazolium bromide solution (0.5 mg mL^–1, 20 μL per well) was added for a final 4 h. Upon completion all solutions were aspirated, dimethyl sulfoxide (150 μl) was added and absorbance (540 nm) was recorded with a Thermo Scientific Multiskan EX microplate photometer.
Oral toxicity was established by feeding cohorts of Manduca sexta³¹ one-day-old neonate larvae with each flexicate (25 μg mL^–1 in artificial wheat germ diet) for 7 d at 28 °C and weighing to assess growth rate. Systemic toxicity assays^{47 (link)} were conducted by injecting an ethanol (70% v/v) swabbed region of first day fifth instar M. sexta larvae with each flexicate (0.5 mg mL^–1 [0.25 μM] in PBS), before allowing them to continue feeding for 7 d at 28 °C, using physical stimulus to assess their status.

Crystal violet assay was used for cell proliferation viability assay. Cells were seeded in 96-well plates at cell-line-specific densities in a complete medium (Supplementary Table S1). The day after, the medium was replaced by fresh medium containing increasing regorafenib concentrations (2 × 10⁻⁶–2 × 10⁻⁵ M). After 72 h of treatment, the medium was removed. The cell layer was gently rinsed with phosphate-buffered saline (PBS) before fixation with 1% glutaraldehyde/PBS for 15 min and stained with 0.1% (v/v in water) crystal violet for 30 min. Finally, cells were washed with running tap water and afterward lysed with 0.2% (v/v in water) Triton X-100 for 90 min. The absorbance was measured at 570 nm using a Multiskan EX Microplate Photometer (Thermo Fischer Scientific, Waltham, MA, USA).

sCD59 concentrations in serum were determined via ELISA (USCN Life Science Inc., China) according to manufacturer’s instructions. Serum samples were thawed, diluted 1:400 in PBS, and incubated on NUNC maxisorp plates (NUNC, Roskilde, Denmark) coated with a mouse anti-human CD59 monoclonal antibody for 2 hours at 37 °C. The plates were washed and incubated with biotin-conjugated rabbit anti-human CD59 polyclonal antibodies for 1 hour at 37 °C. Plates were washed again and incubated for 30 minutes with streptavidin-horseradish peroxidase (HRP). Bound biotinylated antibodies were visualized with TMB substrate for 15 minutes at 37 °C. The reaction was terminated with H₂SO₄. Optical density of the wells was measured at 450 nm with a Multiskan EX Microplate photometer (ThermoScientific, IL). OD450 values were compared to standard concentrations of recombinant CD59 and are expressed in pg/ml. The minimal detectable dose of human sCD59 is 6.7 pg/ml. Intra- and inter-assay coefficients of variation are <10% and <12% respectively. Furthermore, the recovery rate of the ELISA in our hands was 87% (range 83–91%), which was in concordance with the manufacturers description (average recovery rate in serum 87%, range 80–94%). All samples were measured in duplicate.