Spark10m multiplate reader

Keratinocytes were cultured in 12-well plates for 48 hrs in EpiLife medium supplemented with either 100 nM calcipotriol or DMSO and without antibiotics. C. albicans or P. aeruginosa (patient isolate) were grown to early log phase in Luria broth (LB). 35,000 colony forming units/well were seeded into a 96-well black microplate (Greiner Bio-One, Kremsmünster, Austria) and 100 µl of conditioned cell culture medium from untreated or treated cells were added. After 3 hrs incubation at 37 °C, 100 µl of resazurin solution (0.5 nM resazurin, Sigma-Aldrich) was added. Plates were incubated at 37 °C overnight in a Spark 10 M multiplate reader (Tecan, Grödig, Austria) which measured fluorescence at Ex 535 nm/Em 590 nm every hour. C. albicans and P. aeruginosa were treated directly with increasing concentrations of LL-37, 100 nM calcipotriol, or DMSO served as controls.

The cytotoxic effect was studied using the MTT test. Cells were cultivated at a density of 1 × 10⁴ cells/well in a 96-well flat-bottom plate, with three duplicates for each treatment. After 24 h of incubation, cells were treated with varying doses of EO (3.91–1000 µg/mL) for 24 h. To remove extract residues that may interfere with the MTT assay, a wash step with phosphate-buffered saline 1× (PBS) was performed. Cell viability was then measured after 5 h of incubation with 100 μL 1 mg/mL MTT. After removing the solution, 150 μL of DMSO was added to dissolve the crystals. The absorbance of viable cells was measured at λ = 570 nm using a SPARK10M multiplate reader (Tecan, Männedorf, Switzerland). Each plate contained a blank control (medium alone) and a positive control (DMSO 100%) [46 (link)].

The effect of L. alpinum callus cultures’ methanolic extract was investigated using the MTT assay. Cells were grown at a density of 1 × 10⁴ cells/well in a 96-well flat-bottom plate, each treatment having 3 replicates. After 24 h incubation, cells were treated with different concentrations of extract (6.25–400 μg/mL) for 48 h. To remove possible extract residues that might interfere with MTT assay, a wash step with Phosphate Buffer Saline 1× (PBS) was performed, then cell viability was determined after incubation for 3 h with 100 μL 1 mg/mL MTT. Afterwards, the solution was removed and 150 μL of DMSO were added to dissolve the crystals. The absorbance corresponding to the viable cells was measured at λ = 570 nm on a SPARK10M multiplate reader (Tecan, Männedorf, Switzerland). A blank control (medium only), and a positive control (DMSO 100%), were included on every plate. The experiment was repeated three times. Statistical analysis was performed using Graphpad Prism (version 8) software (GraphPad Software, San Diego, CA, USA). Cell viabilities were expressed as percentage of control (set as 100%) and represented as mean ± SEM. Data was analyzed by one-way ANOVA followed by Dunnett’s multiple-comparison test.