Columbia blood agar plates

Blood samples were donated by healthy volunteers who had undertaken informed consent in accordance with local Research Ethics Committee approval. Peripheral blood mononuclear cells were isolated from citrated peripheral blood samples by density gradient separation using Lympholyte (Cedarlane Labs), and subsequent CD14⁺ positive selection using the MACS Miltenyi Biotec Human CD14 microbead protocol (Miltenyi Biotec). CD14⁺ cells were differentiated into macrophages using recombinant human granulocyte-macrophage colony-stimulating factor (200 ng/ml GM-CSF) and recombinant human interferon gamma (50 ng/ml IFN_γ) (Peprotech) in standard tissue culture DMEM media containing fetal calf serum, penicillin and streptomycin. Following removal of antibiotics, macrophages were infected at a multiplicity of infection of 10:1 with M. abscessus 19977 for 2 h, washed in sterile phosphate buffered saline, and then incubated in DMEM media with FCS and 25 μM of compound for 24 and 48 h. At the given time points, supernatant was saved for cell cytotoxicity studies, and M. abscessus survival within the macrophages calculated by macrophage lysis in sterile water, and colony forming unit calculation on Columbia Blood Agar plates (VWR BDH).

The hemolytic potential of the protein and peptide variants was tested on Columbia blood agar plates (VWR). Sterile filter discs (Ø6 mm) were placed on the agar plates and loaded with 20 μg (∼10 μL) of each protein and peptide. Sterile water and 20% Triton-X 100 served as negative and positive control, respectively. The plates where incubated for 24 h at 37°C before evaluation.
AMP toxicity was determined on HKC and HDF. Keratinocytes and fibroblasts were isolated and grown in CnT-07 (CellnTEC) or R10 (Supplementary Table S2), respectively, as described previously (Blunder et al., 2017 (link)). Fluorescence viability staining was performed on HKC and HDF cells grown on chambered cell culture slides (Falcon). 4 × 10³ cells per well were seeded and grown at 37°C and 5% CO₂ before AMPs were added at 30 μM for 24 h. Cells were washed with PBS and the fluorescent dyes PI (1 μg mL^-1) and Hoechst 33342 (20 μg mL^-1) were added for 10 min in the dark. The cells were washed three times with PBS and observed with a Zeiss Axioplan fluorescence microscope (Zeiss), equipped with an Axiocam 503 mono microscope camera (Zeiss), excitation/emission filters 365/420 nm for blue fluorescence, and 546/590 or 565/620 nm for red fluorescence. Image acquisition and editing were done with ZEN 2 (blue edition) microscope software (Zeiss) and GNU Image Manipulation Program (GIMP 2, version 2.8.10).