Genios microplate reader

MSCs from different sources (N = 3 in duplicates for each of MSC isolation source) were expanded for 5 days in CCM and treated for 2 hrs with mitomycin C (10 µg/ml) at 37 °C to arrest cell proliferation. After extensive washing with CCM, the medium was changed either to CCM or PL-free medium without any supplements. MSCs were cultured for 24 hrs, followed by the addition of Alamar Blue (AB) to reach a10% concentration. After 4 hrs of additional incubation, the fluorescence level of the reduced AB was assessed using a TECAN GENios microplate reader (Tecan) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm⁶⁵ . The ratio between the fluorescence of experimental and blank sample (same medium without cells) was used as the AB value. The data was presented as relative fluorescence units (RFU).

To evaluate the metabolic activity of NSCs, 24 h after Rapa treatment, culturing in 10% Alamar blue (7-Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt) (AB) (Sigma-Aldrich, St. Louis, MO, United States) in NSC medium was followed. For this experiment, cells were seeded into 24-well plates at approximately 145,000 cells per well, with 4 technical replicates for each time point. After 3 h of further incubation, the fluorescence level of reduced AB was evaluated using a TECAN GENios microplate reader (Tecan, Männedorf Switzerland) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm (Petrenko et al., 2005 ). The ratio between the fluorescence of the experimental and blank samples (the same medium without cells) was used as the AB value. Data were presented as relative fluorescence units (RFU). Values for treated samples were normalized to those for time-matched controls.

Cell cytotoxicity was measured by determining membrane integrity of HaCaT cells following treatment with crude extracts or purified phytochemicals by means of the CytoTox-ONE™ Assay according to the manufacturer’s instructions (Promega, WI, USA). In brief, cells were plated at 2 × 10⁴ cells/well in 96-well plates and incubated under the above-mentioned conditions for 24 h. Extracts or pure compounds at different concentrations were added to the cells and left to incubate for 24 h at 37 °C and 5% CO₂. After incubation, the assay plates were transferred to 22 °C for 5 min, 100 μL of the CytoTox-ONE™ reagent was added to all wells and incubated at 22 °C for 10 min. After that, 50 μL of stop solution was added to all wells and plates were shaken at 500 rpm for 10 s. The fluorescence signal was measured with an excitation wavelength of 560 nm and an emission wavelength of 590 nm with the Tecan GENios Microplate Reader (Tecan Trading AG, Männedorf, Switzerland). Withaferin A was used as positive control. The triplicate wells without cells were used as negative control to determine background fluorescence. Vehicle control was triplicate cells with untreated cells, the same solvent used to deliver the test compounds. In addition, 2 μL of lysis solution was used as maximum LDH release control.

After incubation of cells with Si substrate and PANI-IO coating for 24 h, cell viability was evaluated by MTT test. For this purpose, the culture medium was discarded, and Caco-2 cells were incubated with 500 μL of a 1 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (M5655, Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. Afterward, the MTT solution was removed, and the formazan crystals that formed as a result of mitochondrial dehydrogenase activity were solubilized with 250 μL isopropanol. Finally, the optical density was read with a Tecan GENios microplate reader (Tecan Trading AG, Männedorf, Switzerland) using a 595 nm wavelength. The results were expressed in percentages related to control (100% viability).

Cell viability of HGF-1 cells was evaluated using the MTT assay based on the reduction by mitochondrial dehydrogenases of the tetrazolium dye MTT to a violet formazan. The cells were seeded in 96-well plates (3.25 × 10⁴ cells/mL), incubated overnight, and then treated with HAp and ZnHAp suspensions. After 24 and 72 h of incubation, the medium was completely aspired, the cell monolayer was washed with phosphate buffer saline (PBS), and incubated with a 1 mg/mL MTT solution for 2 h, 37 °C, in the dark. Afterwards, the MTT solution was discarded and the formazan crystals were dissolved in 120 µL isopropanol. Coloration intensity was estimated by absorbance spectrophotometry at a wavelength of 595 nm, using a Tecan GENios microplate reader (Tecan Trading AG, Männedorf, Switzerland). The results were calculated as percentages (%) of control (100%, untreated cells).

Mice were anesthetized and blood were obtained (300µl) by puncture of the vein of the tail. Serum was stored at -20ºC until assay. Serum from Control (n=16) and K5-CYLD^C/S (n=16) mice of 15-20 months of age were analyzed for the expression of both TNF-α and IL-6 cytokines (LEGENDplex Multi-Analyte Flow Assays Kit, Biolegend). Cytokines were measured in a Tecan GENios Microplate Reader (Tecan Trading AG, Switzerland).

10³ cells were seeded in sextuplicate in 96-wells one day prior to addition of following reagents: 0.5 μg/ml Dox, 10 ng/ml human recombinant TGF-ß1 and 100 ng/ml human recombinant GDF-15. After 6 days, MTS proliferation assays was performed with the CellTiter 96 Aqueous kit (Promega, Madison, USA) according to the manufacturer‘s instruction. Spectrophotometric measurements were conducted using the GENios microplate reader (Tecan Group Ltd., Männedorf, Switzerland) and the absorbance value of the cell-free medium was substracted from the absorbance values of the samples.