Nunc lab tek chamber slide system

ASF-4 cells were seeded in a 4-chamber slide (Nunc™ Lab-Tek™ Chamber Slide System, Thermo Fisher Scientific Inc., Waltham, MA, USA) at a concentration of 0.5 × 10⁵/mL in each chamber on the day before exposure to GJ-EVs. GJ-EVs were dyed with SYTO^R RNASelect^TM green fluorescent cell stain (Invitrogen, Carlsbad, CA, USA) for 3 h and then observed with a laser microscope (Leica TCS SP8, Leica MYCROSYSTEMS, Wetzlar, Germany). PBS was used instead of EVs for control samples.

The kinetics of the hyphal growth was examined by time-lapse video microscopy, as previously described [42 (link)]. In brief, 10⁵ swollen conidia/mL were transferred to the 8-well Nunc™ Lab-Tek™ chamber slide system (Thermo Scientific, Vienna, Austria), and live imaging was performed in a cell incubator at 37 °C and 5% CO₂ using an ioLight Portable Microscope (ioLight, Hampshire, UK) operated with the ioLight app (Vers.: 1.1.1.483) at 37 °C. Video sequences, tracking and quantification of the velocity of hyphal growth were prepared using Fiji software [48 (link)] and the “Manual Tracking” plug-in (developed at Institute Curie by F. Cordeli for ImageJ; Orsay, France). Figures were prepared using Fiji software (based on ImageJ 1.51n, maintained by the Laboratory for Optical and Computational Instrumentation at the University of Wisconsin-Madison, Madison, WI, USA) and Adobe Photoshop (Adobe Systems Incorporated, San José, CA, USA).

General supplies, reagents, and growth media were purchased from Fisher Scientific (Hampton, NH). An 8-ply 100% cotton gauze was purchased from Kendall Curity®; Coviden (Mansfield, MA). Five clinically-relevant topical products were purchased via the pharmacy at the Department of Veterans Affairs in Salt Lake City: gentamicin sulfate ointment USP, 0.1% (Perrigo Company, Allegan, MI), mupirocin ointment USP, 2% (Glenmark Pharmaceuticals, Mahwah, NJ), silver sulfadiazine cream, USP 1% (Ascend Laboratories, Montvale, NJ), Neosporin® (400 U Bacitracin Zinc– 3.5mg Neomycin Sulfate– 5,000 U Polymixin B Sulfate; Johnson and Johnson, New Brunswick, NJ), Altabax® (retapamulin ointment) 1%, (GlaxoSmithKline, Barnard Castle, County Durham, United Kingdom). Hyaluronic acid (HA; 1.01 MDa– 1.8 MDa) was purchased from Lifecore Biomedical (Chaska, MN; catalog #HA15M-5). This HA is a bacterial fermentation product of Streptococcus pyogenes. Reagents and chemicals for synthesizing CZ-01179 were purchased from Sigma Aldrich (St. Louis, MO). Cellulose discs (6 mm) were purchased from BD (Sparks, MD), and collagen coupons were cut from HeliPlug® Collagen Wound Dressing (Integra LifeSciences, Plainsboro, NJ). BacLight™ Bacterial Viability kits were purchased from Molecular Probes (Eugene, OR). The Nunc™ Lab-Tek™ chamber slide system was purchased from ThermoScientific™ (Waltham, MA).

MCF-7, HCT116, and HeLa cell lines were seeded in 8-well Nunc™ Lab-Tek™ Chamber Slide System (Thermo Fisher Scientific) at 35 × 10⁴ cells/well together with 0.25, 0.5, and 1 mg/mL of each β-Nb₂ZnO₆ (A) and (B) nanoparticles for 48 h. The cells were fixed using cold absolute methanol for 10 min, then treated with ProLong™ Gold Antifade Mountant plus DAPI (4′,6-diamidino-2-phenylindole) from TFS. The illustrations were recorded with an LSM 700 confocal microscope (Zeiss, Jena, Germany).

Primary cultures of sensory neurons in the DRG of adult mice were prepared as previously described (5 (link)). Briefly, DRG from the cervical to the lumbar level were dissected and incubated with 2.0 mg/mL collagenase (CLS-3, Worthington Biochemicals) and 50 U/mL dispase (354235, Corning) and subjected to density gradient centrifugation (5 minutes, room temperature, 200g) with 30% Percoll PLUS (28-9038-34 AA, GE Healthcare Bio-Sciences Corp.) to eliminate the myelin sheath. Dissociated DRG neurons were suspended in DMEM: nutrient mixture F-12 (DMEM/F-12) (Fujifilm Wako Pure Chemical Corp.) with 10% FCS and 1% PS and seeded onto 10 μg/mL poly-L-lysine–coated (P1524, Merck) and 10 μg/mL laminin-coated (120–05751, Fujifilm Wako Pure Chemical Corp.) wells of 8-well chamber slides (Nunc Lab-Tek Chamber Slide System, 177402PK, Thermo Fisher Scientific). After being incubated in serum-containing medium for 16 hours, the neurons were maintained for 24 hours (for coculture experiments) or 32 hours (for monoculture experiments) in DMEM/F12 with 2% B-27 (A3653401, Thermo Fisher Scientific).