Synergy 4 fluorescence microplate reader

Polymerization of 2 μM rabbit skeletal muscle G-actin (5% pyrene-labelled) was initiated with 1 × KMEI-polymerization buffer (50 mM KCl, 1 mM MgCl₂, 1 mM EGTA and 10 mM imidazole, pH 7.0) and in the presence of various concentrations of FMNL or Drf3 fragments with or without 10 μM PFN, and was monitored in 96-well plates by a Synergy 4 fluorescence microplate reader (Biotek). Maximum slopes of polymerization curves were determined by linear regression and averaged, n=4. The slopes were normalized and plotted against the concentration of the respective formin construct.

BODIPY-TR-cadaverine (Life Technologies) was prepared in 10 mM NaCl or 5 mM HEPES (pH 7.5) and pre-warmed at 37°C. Purified P. aeruginosa LPS and S. aureus LTA (Sigma-Aldrich) were prepared in distilled water. Colistin and meropenem (Santa Cruz Biotechnology) were used as positive and negative controls respectively (Ouberai et al., 2011 (link)). A final 100 μl mixture of 5 μM BODIPY-TR-cadaverine (in 10 mM NaCl or 5 mM HEPES, pH 7.5), 15 μg/ml LPS or LTA, and peptides or antibiotics in a concentration ranged from 0.02 to 272 μM was added to a 96-well black assay plate. The fluorescence at 580/620 nm was measured at 37°C with a Synergy 4 fluorescence microplate reader (BioTek). Displacement percentage of KAMP relative to colistin and meropenem was determined by: fluorescence of (probe/endotoxin/KAMP mixture - probe/endotoxin/meropenem mixture) / fluorescence of (probe/endotoxin/colistin mixture - probe/endotoxin/meropenem mixture) × 100%.