Alamar blue

Cytotoxicity was measured using the 3D EpiDerm skin tissues and HeLa (ATCC CCL-2) cells using a fluorescent reporter of cell viability, alamarBlue (ThermoFisher Scientific). 3D EpiDerm skin tissues (MatTek EPI-200-AFAB) were transferred to a 12-well plate containing 600 μL of AFAB-assay medium supplemented with 10% vol/vol alamarBlue. STIP3-29 (250 μg/mL; 40-fold MIC) or PBS was added to the apical surface of the tissues and incubated at 37°C in a humidified 5% CO₂ environment for 24 h. HeLa cells were seeded in a 96-well plate at 2 × 10⁴ cells/per well and incubated overnight at 37°C in a humidified 5% CO₂ environment to prepare wells at ∼50% confluence for treatments after spent Dulbecco’s modified Eagle’s medium (DMEM) was removed and cells were washed with PBS. Assays were then performed using phenol red-free DMEM supplement with 9% vol/vol alamarBlue and peptide concentrations varying from 3.13 to 200 μg/mL. After a 24 h incubation period, alamarBlue fluorescence emission was measured at 590 nm upon excitation at 560 nm using Varioskan Lux plate reader (Thermo Scientific). Fluorescence intensities of treated cultures were normalized to measurement made for untreated control samples. Experimental data sets were fit to nonlinear dose-response curves to determine IC₅₀ values using Origin 2019.

Each drug was assayed at six concentrations. Each cell line was seeded on two 384-well plates with 4,000 cells per well and four replicates per plate. Each plate included a control for background fluorescence signal, 10% dimethyl sulfoxide (DMSO), and another control for drug vehicle, either water or DMSO at 0.1%, with the exception of temozolomide, which had DMSO at 0.08%, 0.20%, 0.41%, 0.62%, 0.82%, and 1.23%. Each cell line was incubated for 72 h with each of the treatments for all tested concentrations, dyed with alamarBlue (BioSource International), and incubated for another 24 h. The alamarBlue assay is a fluorometric and colorimetric cell viability assay incorporating an oxidation-reduction indicator that responds to cellular metabolic reduction, with the intensity of fluorescence produced proportional to the number of living cells. Subsequently, a Tecan Freedom EVO 150 robotics system with a Connect stacker and F200 plate reader, which measures fluorescence intensity in raw fluorescence units (RFUs), was used for viability measurements. We applied quality control procedures and calculated cell viability as previously described [1 (link), 22 , 29 (link)].

Biocompatibility and cell attachment to the mesh were evaluated by seeding HDFs (10,000 cells/well) onto ethanol-sterilised mesh samples (15.6 mm diameter). The seeding efficiency of LCM1 and LCM2 was 10 and 5%, respectively (data not shown). Samples were analysed at 3, 7, 14 days.
Proliferation of HDFs seeded on the meshes was evaluated with alamarBlue (Invitrogen, Thermo Fisher), with 1:9 ratio (alamarBlue to growing media ratio). Control samples were HDFs cultured in DMEM supplemented with FBS and P/S. Samples were incubated for 4 h at 37 °C, 5% CO₂ and transferred to a 96-well plate (triplicates of 100 µl from each sample). Samples were imaged with a CLARIOstar^® microplate reader (BMG LABTECH GmbH, Germany) in fluorescence mode (excitation 560 nm, emission 590 nm).
Samples of the seeded meshes were fixated in 4% paraformaldehyde for 30 min and washed with Phosphate Buffer Solution (Thermo Fisher Scientific). After permeabilization using 0.25% Triton X-100 (Sigma Aldrich), samples were washed in PBS and stained using Phalloidin TRITC (Sigma) 1:1000 in PBS for 1 h to visualise cells’ actin filaments. Following these steps, the samples were mounted on glass microscope slides with VECTASHIELD^® DAPI (Vector Laboratories Inc.) to visualise nuclei. Samples were imaged with Zeiss AxioObserver Microscope with ApoTome.2 feature and Zeiss ZEN software (Zeiss, Oberkochen, Germany).