Lt 4000 microplate reader

Here, we determined the optimal seeding density of A. castellanii trophozoites that were used in testing the anti-acanthamoebic activity of C. sinensis. Briefly, A. castellanii trophozoites at various numbers (1 × 10³, 2.5 × 10³, 5 × 10³, 7.5 × 10³ and 10 × 10³) were seeded in a volume of 100 μl PYG medium/well of 96-well plates. Control wells included only 100 μl PYG medium. The culture plates were checked under an inverted microscope (CETI, UK) to ensure the presence of trophozoites, and incubated in a Stuart oven at 25 °C. After 24, 48 and 72 h, the plates were fixed and stained using the SRB assay as described above and strictly according to the protocol adapted for A. castellanii by Ortega-Rivas et al. (2016) (link). The OD of each well was determined by measuring the colour absorbance spectrophotometrically at wavelengths of 450, 492 and 630 nm using an L-T 4000 microplate reader (Labtech, UK). The OD values of each seeding number were compared to the other seeding numbers and to the blank control at each of the examined time points (i.e. 24, 48 and 72 h).

hMSCs, previously treated with morphine sulphate, were seeded in a 96 well plate at the density of 1 × 10⁴ cells/well in the same experimental conditions as previously described. At the end of the treatment, the medium was changed with a new one containing 0.5 mg/mL of b3-(4,5-dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA), for 3 h at 37 °C in 5% CO₂. The medium was then discarded and the resulting formazan crystals were dissolved by adding dimethylsulfoxyde in isopropanol (1:1). Colorimetric reaction was finally quantified by microplate reader (LT-4000 Microplate reader, Labtech L.t.d, Heatfield, UK), measuring the absorbance at 578 nm and a reference wavelength of 690 nm.

Cells were seeded in 96-well plates at a density of 10⁴ cells per well and treated with TRAIL and/or bortezomib after 24 h. For inhibitor studies, cells were pre-treated for 1 h with 20 μM zVad and 50 μM nec-1 was included in the treatment. At the indicated times, 20 μl MTT reagent (5 mg/ml; Calbiochem, Watford, UK) was added, followed by incubation for 2–3 h and addition of 150 μl MTT solubilisation solution (50% dimethylformamide, 0.2% glacial acetic acid, 20 mM HCl, 10% SDS). After incubation overnight, the OD₅₉₅ was measured on a LT-4000 microplate reader (Labtech, Uckfield, UK).
Statistical analysis of the results from at least three independent experiments as indicated was performed using GraphPad Prism 6 software (Graphpad Software Inc., La Jolla, CA, USA) and one- or two-way ANOVA as appropriate and specified. Drug synergy was determined using CompuSyn software (ComboSyn Inc., Paramus, NJ, USA) according to the Chou-Talalay method.^{51 (link)}

Briefly, 5 × 10⁴ Vero cells grown in 96-well plates in MEM were treated with riboflavin at a concentration range from 1000 to 0.48 µM for 48 h. Then, 1 mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, Aldrich, Saint Louis, MI, USA) was added to the cells, and they were incubated for 1h. Formazan crystals were dissolved in DMSO, and absorbance was determined at 550 nm using a microplate reader spectrophotometer (LT-4000 Microplatereader, LabTech). The results are shown as the percentage of viable cells relative to the untreated control cells. All assays were performed three times independently in quadruplicate. From this, the CC₅₀ (cytotoxic concentration of the compound that reduced cell viability to 50%) was calculated from a dose–response curve in GraphPad Prism (version 8.00), using four-parameter curve-fitting.

Cell proliferation was assayed by BrdU assay kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, sf-MSCs, isolated from healthy and OA synovial fluid, were seeded into a 96-well culture plate with MEM containing 10% FBS at the density of 8 × 10³ cells/well. After 24 h, the medium was changed with a fresh one containing 10 μM BrdU for 48 h at 37 °C. After the removal of the labeling solution, cells were fixed and incubated with anti-BrdU conjugated with peroxidase antibody for 90 min at RT. After three washing steps in PBS, a tetramethyl-benzidine (TMB) substrate solution was added for 10 min at RT, and the reaction was stopped with 1 M H₂SO₄. The optical density was measured using a spectrophotometer microplate reader (LT-4000 Microplate reader, Labtech Ltd., Heathfield, UK) at a wavelength of 450 nm and a reference wavelength of 690 nm. Data were expressed in optical density (O.D.) and were represented as mean values ± SD. Results of each donor were shown. The experiments were repeated at least four times, and each experimental point was done in triplicate.

Tetrazolium bromide (MTT) assay was used to assess cell viability. vMSCs were seeded in triplicate (one triplicate for each morphine concentration) into a 96-well culture plate at the density of 8 × 10³ cells/well for 24 h. Then, the medium was changed with a fresh one containing the morphine sulphate solutions to be tested.
After 7 days, 10 µl of 5 µg/ml of MTT (Sigma-Aldrich, St. Louis, MO, USA) was added in each well and incubated for 3 h at 37 °C in 5% CO2. Formazan crystals were dissolved by adding dimethylsulphoxide (DMSO) in isopropanol (1:1). Optical density was measured, using a spectrophotometer microplate reader (LT-4000 Microplate reader, Labtech L.t.d, Heatfield, UK) at 578 nm (reference wavelength 690 nm).
Results were expressed in percentage as relative viability compared to control samples.