Plate thermoshaker

To prepare seeds, FVIII samples (0.122 μM) were incubated for 2, 5, 8 or 18 h at 45°C in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan). Native FVIII samples (0.122 μM) were mixed 1:1 with the corresponding seeds and time-dependent aggregation at 45°C was initiated. Samples were stored at −80°C until HPLC-SEC analysis.

Bacterial test cultures were grown in the Mueller-Hinton broth (MHB, Sigma, St. Louis, MO, USA) at 37 °C to mid-log phase and then diluted with the 2× MHB media supplemented with 1.8% NaCl or without salt to reach a final cell concentration of 10⁶ CFU/mL. An amount of 50 µL of the obtained bacterial suspension was added to aliquots of 50 µL of the peptide solutions serially diluted with sterilized 0.1% bovine serum albumin (BSA) in 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011, Hamburg, Germany). After incubation for 24 h at 37 °C and 950 rpm on the plate thermoshaker (Biosan, Riga, Latvia), minimum inhibitory concentrations (MIC) were determined as the lowest peptide concentrations that prevented growth of a test microorganism observed as visible turbidity. In most cases, no significant divergence of MIC values was observed (within ±1 dilution step). The results were expressed as the median values determined on the basis of at least three independent experiments performed in triplicate.

The antimicrobial activity of investigated peptides was determined by the method of two-fold serial dilutions in the sterile 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011, Hamburg, Germany). Mueller–Hinton broth was used as a culture fluid. The bacterial strains were cultured in the MH medium at 37 °C for 16 h, then aliquots of culture were diluted with fresh MH and grown to optical density (OD₆₀₀) of 1. The bacterial cultures were prepared in the NaCl-enriched MH broth (42 g/L Mueller–Hinton broth, 18 g/L NaCl) to the final concentration of 1 × 10⁶ colony-forming units (CFU)/mL. In the next step, 50 μL of obtained cell culture was added to 50 μL of peptides to reach the final cell density of 5 × 10⁵ CFU/mL; the compounds had been previously diluted with sterilized 0.1% BSA. After adding the suspension of cells, the plate was incubated at 37 °C for 24 h and 1000 rpm on the plate thermo-shaker ((Biosan, Riga, Latvia). The values of MIC (minimum inhibitory concentration) were identified as the lowest concentration of peptide at which the growth of the culture had not taken place and defined visually, or 0.1 mg/mL of resazurin was added to each well and incubated at 37 °C for 2 h. The tests were carried out at no fewer than two repetitions of free independent experiments, and the MIC of peptides was calculated as an averaged value of these assays.