12 well plate

To package lentivirus, the library plasmid was co-transfected with pMD2.G and psPAX2 into 120 million HEK293FT cells, using Lipofectamine 2000 (Thermo Fisher) in 10 T-225 flasks (Sigma). After 72 h, the supernatant was harvested and concentrated according to the LentiX Concentrator protocol, then stored at −80 ^oC. In order to titer this lentiviral preparation, we spinfected 3 million HEK293T cells per well with 0, 40, 80, 120, 160, or 200 µl of the lentiviral supernatant in 2 ml D10 media supplemented with 8 µg/ml polybrene (Sigma) in 12-well plates (Sigma). The plates were spun at 1000 × g for 2 h at 37 ^oC. After spinning, 2 ml fresh D10 media was added to each well and the cells were maintained at 37 ^oC for 24 h. The cells were then dissociated with TrypLE, suspended in D10, and counted with a TC20 Automated Cell Counter (Biorad). Then, 2500 cells per well were plated in a black TC-treated 96-well plate with clear well bottoms (Sigma). For each dose, four wells then underwent puromycin selection while four wells continued growth in D10 media. Media was refreshed after 48 h. After 96 h, survival of the selected cells in comparison to the unselected cells was measured by CellTiter-Glo (Promega) and used to calculate the lentivirus MOI. The SaCas9 lentivirus was similarly prepared and titered, using blasticidin selection.

Upon reaching 80% confluency, human astrocytes in 12-well plates (Merck Sigma) were subjected to OGD for 8 h. The media were subsequently replaced with 0% FBS-containing DMEM (HyClone). Various concentrations of KRGE were added, and the cells were incubated for 23 h. Thereafter, 30 μL of a cell-counting kit-8 (CCK-8) agent (Dojindo, Fukuoka, Japan) was added to each well, and cells were incubated at 37 °C for 1 h. We used a plate reader (Epoch Microplate Spectrophotometer; BioTek, Santa Clara, CA, USA) to determine the absorbance at a wavelength of 450 nm. The background wave was determined at 630 nm and subtracted from the value measured at 450 nm.

PCL with molecular weight (M.W.) 80,000 g·mol⁻¹, methanol, chloroform, GEN, Mueller Hinton Agar, 12-well plates, 96-well plates, and Petri dishes were acquired from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was obtained from Honeywell, Fluka, Seelze, Germany. The bacterial strains E. coli ATCC 25922, S. aureus ATCC, B. cereus ATCC 11778, and P. aeruginosa ATCC 10,145 were obtained from the American Type Culture Collection (ATCC). The culture medium DMEM/F12 and the culture medium supplemented with 10% fetal calf serum, streptomycin, and penicillin were acquired from Gibco LifeTechnologies, Paisley, UK. All commercial materials were used without further purification.

Thermal damage was created by burning 10-day air–liquid interface skins at 100 °C for 4 s with a digital soldering iron (FX888D, American Hakko Products, Inc., Santa Clarita, CA). The skin filter inserts were transferred to 12-well plates (Sigma-Aldrich) containing 800 μl/well fresh culture medium prior to bacterial infection. MRSA biofilm was established by seeding 2 × 10⁶ CFU in 5 μl PBS on top of the thermal damaged skin and cultured at 37 °C and 5% CO₂ for 24 h. DJK-5 peptide (30 μl of 0.4%) alone or in combination with 0.01%, 0.1%, or 0.4% 1002 was administered on top of the pre-formed biofilm. Skin samples were collected 24 h post peptide treatment for colony count.

Zebrafish were raised and kept at 28°C under a 14 h light/10 h dark cycle with twice-a-day feeding at the Radboud University Zebrafish Facility. The mutant line fgf8a^ti282 (ace) was obtained from the European Zebrafish Resource Center (EZRC, Karlsruhe Institute of Technology, Germany). To obtain homo- and heterozygous mutant embryos, fgf8a^ti282 heterozygous adult carriers were pair-wise crossed. Every 2 weeks, breeding tanks were set up the day before mating, with males and females separated by a transparent wall. The following morning, the wall was removed and the water was changed for low-conductivity warm water of around 29°C to induce spawning. Eggs were collected approximately 60 min after spawning and transferred to petri dishes with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, 0.00001% Methylene Blue). Petri dishes with eggs were placed in an incubator at 28.5°C with a 14 h light/10 h dark cycle. E3 medium was refreshed at days 1, 4 and 5; any unfertilized eggs, dead embryos and post-hatching chorions were removed immediately. At 4 dpf, larvae were anaesthetized with 2-phenoxyethanol (1:750) and a tail clip biopsy was taken from each larvae as previously described (Wilkinson et al., 2013 (link)). Upon tail clipping, the larvae were individually housed in 12-well plates (Sigma-Aldrich, USA).

For osteogenic and adipogenic differentiation, cells were seeded (osteogenic, 20,000 cells/well and adipogenic, 40,000 cells/well) onto 12-well plates (Sigma-Aldrich) and cultured in CM for 3 days under standard culture conditions. The medium was then changed to either StemPro™ osteogenesis differentiation medium (Thermofisher Scientific) or StemPro™ adipogenesis differentiation medium (Thermofisher Scientific). Cultures were maintained for 21 days under standard culture conditions with medium changes every 2–3 days. For chondrogenic differentiation, 500,000 cells were resuspended in 1.5 ml Eppendorf in StemPro™ chondrogenesis differentiation medium (Thermofisher Scientific) and centrifuged for 5 min at 783g. Pellets were cultured for 21 days under standard culture conditions with medium changes every 2–3 days. Negative control samples of each cell type were maintained in CM. After 21 days, cells were fixed in 4% paraformaldehyde (PFA, Alfa Aesar, Haverhill, MA, USA) for 30 min. Osteogenesis and adipogenesis were confirmed using 2% Alizarin Red S and 0.3% Oil Red O, respectively. A 1% Alcian blue solution prepared in 0.1 N hydrochloric acid (HCl) was used to confirm the presence of glycosaminoglycans (GAGs) in chondrogenic differentiation. Stains were visualised using phase-contrast AmScope MD35 microscope (AmScope, Irvine, CA, USA).