96 well tc treated microplate

Cell density and mCherry fluorescence was measured with the POLARstar omega microplate reader and analysed with BMG Labtech Mars Data Analysis Software (Ortenberg, Germany). Cell density was measured in Corning 96 Well TC-Treated microplates at 600 nm. mCherry fluorescence was measured from a Nunclon Surface black F96 microtiter plate with top optics using an Ex584 excitation filter and 600–680 emission filter.
Raw data was blank corrected, subtracting the mCherry fluorescence reading of the liquid the cell culture was suspended in, to remove background fluorescence. This figure was then divided by the cell density (OD₆₀₀) reading for the culture.

The toxicity effects of all the amyloid fibrils were tested on the SH-SY5Y human neuroblastoma cell line using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) colorimetric assay. The cells were grown in 1:1 mix of Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 growth media with 5% Fetal Bovine Serum (FBS) (Gibco) containing L-Glutamine and Phenol Red in 75 mm³ T-75 flasks at 37 °C in 5% carbon dioxide (CO₂) environment till they achieved 70–90% confluency. After 24 h, approximately 2,500 cells were transferred into each reaction well of 96 well transparent tissue culture plate (Corning^® 96 Well TC-Treated Microplates). Once the cells were stabilized, 20 µl of samples were added in each well containing 100 µl culture media. Before the fibrils were added to the cells, they underwent a washing protocol to remove excess/unbound drug compounds as described previously (Palhano et al., 2013 (link)). The samples were added into the cell assay and incubated for 20 h to test their toxic effects. After incubation, 10 µl of the stock MTT reagent (5 mg/ml) was added to each well. After 4 h of conversion into the formazan product, DMSO was added to dissolve the purple crystals left in the dark for 1 h before the measurement of absorbance at 570 nm with 630 nm as a subtracted reference wavelength.

Pure compounds were assayed against the human hepatoma cell line HepG2 (ATCC HB-8065) in an MTT test as 20-point curves with 1:2 dilutions starting at 40 μg/mL in triplicate. Cells were seeded at 10.000 cells/well in a 96-well plate (Corning 96-well TC-treated microplates) for 24 h and after addition of compounds 1–3 and 5 plates were incubated for 72 h. MMS (methylmethanesulfonate, Sigma-Aldrich, 4 mM) was used as the positive control, and DMSO 0.5% as the negative control. After addition of MMT dye (thiazolyl blue tetrazoliumbromide, ACROS Organics), cells were incubated for 2–3 h and supernatant was removed. Resulting formazan crystals were finally dissolved by means of 100 µL DMSO (100%) and absorbance was measured at 570 nm. The obtained data was analyzed using Genedata Screener software (Genedata, Inc., Basel, Switzerland) and Prim 9.4.1 software, from GraphPad was used for titrations representation [57 (link)].

Proliferation assays were performed using the IncuCyte live cell imaging system (Essen Biosciences, Australia). This system enables real-time cell counting by analyzing the occupied area (% confluence) of cell images over time. Briefly, 5 × 10³ cells were trypsinized and seeded in a Corning® 96 Well TC-Treated Microplates in 150 μL media and grown for 24 h. Next day, cells were treated transiently with RES and RES loaded MSNs (10, 15, and 20 μM) and placed in IncuCyte live cell imaging system as described previously (Matin et al., 2019 (link)). Two images per well were taken every 2 h for five consecutive days and confluency of the cells was measured. Each experiment consisted of three independent replicates. The cell viability was plotted and analyzed by the One-Way ANOVA test using GraphPad Prism software.

KB cells were counted and 1.5 × 10³ cells/well were seeded onto a 96-well flat-bottom plastic culture plate (Corning, 96-well TC-treated microplates). After 24 h of growth at 5% CO₂, 37°C, the culture medium was replaced with 100 μl/well of 5, 15, 25 and 50 μg/ml of ZnO NCs or TNHs and 5, 15, 25 μg/ml EVs treatment solutions. After 24 h treatments, the cell viability was assessed, 10 μl of the WST-1 (CELLPRO-RO Roche) was added to each well and after 2 h of incubation at standard conditions, the formazan absorbance was detected at 490 nm by the Multiskan Go microplate spectrophotometer (Thermo Fisher Scientific) using a 620-nm reference.
Cytotoxicity tests were carried out at least in triplicates and the viability values were normalized to control and expressed as mean ± standard error of the mean (SEM).

The biodegradation of dual-functioning scaffolds was investigated after sample soaking in three different solvents: (i) PBS (phosphate buffer solution, without Ca²⁺ and Mg²⁺, Euroclone, Milan, Italy); (ii) H₂O₂ 1.25 mM in PBS; and (iii) β-N-Acetylglucosaminidase (from Canavalia ensiformis (Jack bean), NAGase, Sigma-Aldrich, USA) 5 U/L in PBS.
In details, weighted circular portions of each scaffold (0.32 cm²) were sterilized through UV irradiation for 24 h and, then, placed in a 96-well plate (Corning^® 96 Well TC-Treated MicroPlates, New York, NY, USA). The scaffolds were soaked in 200 μL of solvent (PBS, H₂O₂ or NAGase) and maintained in incubator (Shellab^® Sheldon^® Manufacturing Inc., Cornelius, OR, USA) (95% air and 5% CO₂ atmosphere) at 37 °C for 7 and 14 days; in order to alter nor the pH neither the enzymatic unit, the solvent was changed every 24 h. After both 7 and 14 days, sample degradation was evaluated in terms of morphological alterations and weight loss. Each condition was investigated in triplicate.