Multiskan spectrum

Cells were seeded in 96-well plates at a density of 8 × 103 cells per well (A2780) or 10 × 103 cells per well (SKOV3) overnight. The cells were first treated with different reagents for the indicated times and then treated with the MTT reagents. After 4–6 h, we measured the absorbance at 490 nm using a Multiskan Spectrum (BioTek, Winooski, VT, USA).

In brief, different concentrations of the peptides and antibiotic were incubated with microorganisms under defined conditions. The microbial suspension was diluted with fresh medium to a concentration of 2 × 10⁶ CFU/mL. Peptides and antibiotic were dissolved in the culture medium and then 50 μL of each peptide and antibiotic solution was mixed with 50 μL of diluted bacterial suspensions to get a final concentration range from 0.8–100 μM in two-fold dilution. The wells not treated with antimicrobial agents served as the positive control and the medium without bacteria inoculation as the negative control. After incubation at 37 °C for 24 h, the absorbance of each well was recorded using a multi-well microplate reader (Multiskan Spectrum, Bio Tek, Winuski, VT, USA) at 600 nm. The lowest concentration at which the peptide inhibited the growth of the bacteria completely was taken as the MIC. MBC were determined by plating 50 μL of bacterial suspensions from the MIC assay on and above the MIC values on TSA. Plates where no bacterial growth was visible after incubation at 37 °C for 24 h were considered to be the MBC.

A2780 cells (8 × 10³ cells per well) and ID8 cells (5 × 10³ cells per well) were seeded into 96-well plates and treated with FCCP and/or cisplatin for 24 h. MTT was added, and cells were incubated for 4 h. Media were removed, and 150 μL DMSO was added to dissolve formazan crystals. The absorbance at 570 nm was measured using a Multiskan Spectrum (BioTek, Winooski, VT, USA).

Growth inhibition kinetics of peptides on S. aureus was determined as described previously [50 (link)]. Aliquots of overnight culture of S. aureus were diluted in TSB broth to obtain the final concentration of 1 × 10⁶ CFU/mL. Peptides was then added into S. aureus culture to the final concentrations of 1/8 × MIC, 1/4 × MIC 1/2 × MIC and 1 × MIC. Sterile TSB acted as a blank control. These plates were incubated at 37 °C. The absorbance at 600 nm (OD₆₀₀) was determined using a microplate spectrophotometer (Multiskan Spectrum, Bio Tek, Winuski, VT, USA) every hour throughout 26 h incubation.

The two-fold dilution method was used to determine the minimum inhibitory concentration (MIC) of the peptides as described previously (Leoni et al., 2017 (link)). S. mutans in the exponential phase was diluted to 2 × 10⁶ CFU/ml in BHI. Fifty microliters of GHc or GHd were prepared as a two-fold serial dilution with water in 96-well microtiter plates. Then, 50 μl of diluted bacterial suspension (final concentration of 10⁶ CFU/ml) was added to each well of the plates. The plates were incubated at 37°C under anaerobic conditions for 16 h. The absorbance at 600 nm was detected with a microplate reader (Multiskan Spectrum, BioTek Inc., USA). Non-treated bacteria and BHI broth medium were used as a negative control and blank, and chlorhexidine was used as the positive control. The MIC was defined as the lowest concentration of GHc or GHd that inhibited bacterial growth. Minimum bactericidal concentrations (MBCs) were determined by plating the bacterial suspensions from the MIC assay wells at the peptide concentrations equal to or higher than the MIC values on solid agars. The lowest peptide concentrations at which there was no bacterial growth on plates after incubation were taken as the MBC (Liu et al., 2017 (link)). Resazurin microtiter assay was also performed as previously described to determine the MIC (Palomino et al., 2002 (link)). All sets were conducted in triplicate.

The antimicrobial activities of the peptides were measured by the two-fold dilution method [32 (link)]. Each strain was inoculated in the medium and cultivated to the logarithmic phase. A series of diluted peptides (concentrations of 3.1–100 μM, 50 μL) were prepared in a 96-well plate (Corning, New York, NY, USA), and the same volume of the bacterial suspension (the final concentration of 1 × 10⁶ CFU/mL) was added. After incubation at 37 °C for 18–24 h, the absorbance at 600 nm was measured by using a microplate reader (Multiskan Spectrum, BioTek, Winuski, VT, USA). MIC was the lowest concentration of peptides that completely inhibited bacterial growth. All tests were performed in triplicate independent experiments.
From the MIC measuring plates, 50 μL of bacteria suspension in the presence of peptides when the concentrations were equal and up to MICs was spread on the BHI agar medium, and incubated at 37 °C for 24 h. The peptide concentration with no bacterial growth was defined as MBC [33 (link)].