Both the antifungal-loaded and bare fiber mats were adhered to microscope cover slips (n=4) and plated onto 24-well Nunc dishes. Human corneal and sclera fibroblasts were grown in HyClone® Dulbecco’s Modified Eagle’s Medium (Thermo Fisher) with 10% fetal bovine serum antimycotic solution containing 500 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate, and 2.25 μg/mL amphotericin B (Sigma-Aldrich). The cells were seeded at a concentration of ~4×104 cells/well and incubated with the fiber mats at 37°C for 24 hours. After equilibrating the plates to room temperature, an equal volume of CellTiter-Glo® reagent was added (Promega Corporation, Madison, WI, USA). The adenosine triphosphate content was determined by recording the luminescence on a Tecan Infinity M200 plate reader as per the manufacturer’s instruction.
Infinity m200 plate reader
Manufactured by Tecan
The Infinity M200 is a multimode microplate reader that can measure absorbance, fluorescence, and luminescence. It is capable of UV-Vis absorbance scanning and endpoint as well as kinetic measurements. The Infinity M200 provides accurate and reliable data for a wide range of applications in life science research and industrial settings.
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2 protocols using infinity m200 plate reader
1
Antifungal Fiber Mats Cytotoxicity Assay
2
Measuring Cellular Metabolism via MTT Assay
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To measure cellular metabolism using the MTT assay [30] , 1 × 10 4 HaCaT cells were seeded per well in 4-well plates (2 cm 2 growth area; SPL Life Sciences). Cells were irradiated (in PBS) as indicated in the results section and cultured for the indicated times in regular growth media. Corresponding controls were kept in PBS for the same time as the irradiated samples. At the time of analysis, the medium was removed and the cells were washed carefully in prewarmed PBS. Then 1 mL fresh complete culture medium, including MTT at a final concentration of 0.5 mg/mL, was added and the cells were incubated for 2 h at 37 °C. Then 250 µL DMSO were added per well and the formazan was dissolved by shaking on an orbital shaker for 10 min at RT, protected from light. Reduced MTT was measured by absorbance at 590 nm with a reference at 620 nm using a Tecan Infinity M200 plate reader in duplicates per well (technical replicates), and quadruplicates (biological replicates) per condition, resulting in eight measurements per sample. The measurements were normalised to the corresponding control cells.
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