4.12. Checkerboard testing for synergy analysis

A checkerboard analysis is conducted to determine the antibacterial potency of the combination of amyloid peptides and α-defensins in comparison to their individual activities.⁹⁷ To quantify the interactions between amyloid peptides and α-defensins, the Fractional Inhibitory Concentration (FIC) index is introduced by the equation:

where A and B are the MIC of each peptide in combination, and MIC_A and MIC_B are the MIC of each peptide alone. The combining effect is interpreted as: synergy, FIC index ≤ 0.5; additive or indifference, 0.5 < FIC index < 4.0; antagonism, FIC index ≥ 4.0.⁹⁸

In detail, in a 96-well plate, the 2-fold serial dilutions of amyloid peptides (at concentrations from 1024 to 4 μg ml⁻¹) were added from columns 2 to 10, while the 2-fold serial dilutions of α-defensins (at concentrations from 256 to 16 μg ml⁻¹) were added from row B to G. Column 12 contains a serial dilution of α-defensins alone, while row H contains a serial dilution of amyloid peptides alone, which are set as controls to determine the MIC value for individual peptides alone (Fig. S12d^†). Assay plates are inoculated with 100 μL bacterial suspensions (5 × 10⁵ CFU mL⁻¹), incubated at 37 °C for 20 h. The amount of growth in each well is quantified by OD₆₀₀ and compared with that in the pure bacteria growth control. The MIC is recorded as the lowest concentration of the peptide that inhibits over 90% of growth.