Spectrostar nano plate reader

Following incubations with test compounds and stimulation agents, cell numbers were
quantified by transferring 100 μl cell suspension from each well to a flat-bottomed,
clear, ninety-six-well plate. A sample of 20 ml of
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt and phenazine methosulfate reagent (Promega) was added to each well and the
plate was incubated for 90 min at 37°C and 5 % CO₂. Following incubation,
absorbance was read at 490 nm on a BMG Labtech Spectrostar Nano plate reader. Viable cell
numbers were estimated from a standard curve determined by haemocytometer counting with
0·02 % (w/v) trypan blue stain.

For the cytotoxicity assays, the cells were cultivated on 96-well plates, at a concentration of 7.5 × 10³ cells/mL in 100 μL of media in each well for two days. After that time, the cell culture media was removed, and the wells were treated with dispersions of each silica-functionalized material from 0.1 µM to 100 µM, for 24 h. After incubation, the dispersions were discarded and replaced by 100 μL of medium, without phenol red and without serum, and 10 μL of a 12 mM MTT (dimethylthiazolyl-diphenyl-tetrazolium bromide) solution was added to each well and mixed. After 3 h of incubation, all the supernatants less 25 μL were removed and 100 μL DMSO was added to each well to dissolve the formazan, leaving it 15 min to react. A negative control of cells incubated with media and without any material, and positive control incubating cells with 1:1 vol of DMSO/media solution were also tested. Cell viability was measured with the absorbance at 570 nm using a SPECTROstar Nano plate reader (BMG Labtech Inc., Cary, NC, USA). IC₅₀ values for materials containing tin have been referred to in terms of the concentration of metal in each material. To assess whether the toxicity is due to the metallodrug or the silica nanoparticles itself, materials of the same concentration of silica were compared with and without the cytotoxic organotin(IV) compound.

The biofilm formation and the viability of the cells were tested after 1,8-cineole treatments (Zhong et al., 2017 (link)). Briefly, 100 μl F solani species complex suspensions (2 × 10⁶ CFU/ml) were added to a 96-well microplate to adhesion for 90 min at 27°C. After pre adhesion, the unattached cells were washed by using sterile PBS, then add 100 μl 1,8-cineole (in the range of 23.05 to 2.88 μg/ml in the above described medium), and incubated at 27°C for 72 h to evaluate the effect of 1,8-cineole on biofilm formation.
The inhibitory effect of 1,8-cineole on F. solani species complex biofilm was evaluated using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (Shanghai yuanye Bi-Technology Co., Ltd. Shanghai, China) reduction assay according to the method described in a previous study (Liu et al., 2017 (link)). After incubation at 27 C for 72 h, the biofilms were washed twice with PBS to remove unattached cells and 200 μl XTT solution containing 1 mM menadione was added. After incubation at 37 C for 2 h in dark, 100 μl of each supernatant were transferred to new 96-well plates and the absorbance of each solution was measured at 490 nm using SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany).

100 μl F solani species complex suspensions and 100 μl 1,8-cineole (in the range of 23.05 to 2.88 μg/ml in the above described medium) were added and incubated at 96-well microplates at 27°C for 4 h. Following the initial incubation, the medium was aspirated and unattached cells were washed by using sterile PBS thrice. Adhesion assay was measured by XTT-reduction assay as described previously (Zhong et al., 2017 (link)). The colorimetric change at 490 nm was measured with a SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany).

The viability of human keratinocytes (HaCaT) was detected after peptide-treatments and compared to the untreated control. Briefly, HaCaT cells were seeded into 96-well micro-plates (10,000 cells/well) then cultured in a 37 °C incubator at 5% CO₂ in 95% humidity. On the following day, the cells were treated with increasing concentrations of NCR335 and NCR169C derivatives. After 48 h treatments, HaCaT cells were washed with PBS (phosphate buffered saline) and incubated for an hour at 37 °C with MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) at 0.5 mg/mL concentration diluted in the culture medium. Formazan crystals were solubilized in DMSO (Sigma-Aldrich, St. Louis, MO, USA) and the absorption was measured at 570 nm in SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany). The experiments were performed at least three times using four independent biological replicates.

A total of 95 µL of cell suspensions at 4 × 10⁴ cell/mL in 1/8AIM medium were added into the wells of microplates and supplemented with 5 µL of two-fold dilution series of the peptides or 5 µL of medium as control. Plates were incubated at 37 °C in 5 % CO₂ level for 72 h and the formed biofilms were washed twice with phosphate buffered saline (PBS) to remove the slightly attached cells. The viability of the biofilm-embedded cells was measured with the XTT reduction assay. XTT was solved in PBS at 0.5 mg/mL concentration and supplemented with 1 µM menadion. After adding 100 µL XTT solution to each well the plates were incubated for 2 h at 37 °C in dark. Subsequently 80 µL of each supernatant was transferred to new 96-well plates and the absorbance was measured at 490 nm using SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany). The experiments were carried in 5 biological replicates in duplicates.