96 well plate

For the in vitro experiments, the cell lines of human lung cancer A549 and H460 as well as normal MRC-5 cells were cultured in DMEM (Sigma, Co., MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified incubator with 95% air/5% CO₂ atmosphere. The relative cell growth rate (%) was measured at 48 h after the treatments using MTT assay according to our previously reported methods (Ji et al., 2014 (link); Liu et al., 2016 (link)). Briefly, cells were planted in 96-well plates (Becton Dickinson, NJ, U.S.A.) at 2 × 10³/well and incubated overnight to allow attachment. The cells in control group (0) were treated with DMSO [0.1% (v/v), final concentration]. The cells were incubated in DMEM medium supplemented with 10% FBS containing different concentrations of TBrC (5–100 μg/mL), or Bay11-7082 (Bay, 0.62 μg/mL, a specific NF-κB inhibitor as the positive control). After 48 h of treatment, the absorbance values in each test group were measured using MTT assay. The relative cell growth (%) was calculated based on the absorbance of sample vs. the absorbance of the control (vehicle). Each experiment was repeated three times.

To generate tissue-engineered cartilage, chondrocytes were cultivated as 3D high-density micromasses in 96-well plates (Becton Dickinson, Heidelberg, Germany). Each well was filled with 6 × 10⁵ chondrocytes pooled from three out of 12 donors (n = 4 donor groups) per 200 µL complete RPMI medium. To achieve ECM build-up, cells were cultivated for 14 days. For the next seven days, medium that was supplemented with 0, 0.05, 0.5, 5, 50, or 500 nmol/L CCL25 was added and changed every 24 h. To simulate OA-like changes, CCL25 treatment was combined with the addition of 0.6 nmol/L TNF-α (R&D Systems, Wiesbaden, Germany) [22 (link)]. Micromasses of all four donor groups were used to investigate the effect of 0–500 nmol/L CCL25 on normal (without TNF-α) or OA-like chondrocytes (with TNF-α).

Cells (5 × 10³ cells per well) were seeded on 96-well plates (Becton Dickinson Labware) in D-MEM/Ham’s F-12 with 10 % FBS and 1 % penicillin/streptomycin. Twenty four hours later, cells were either remained untreated or were treated with any one of the following drugs: 2 μg/ml 5-fluorouracil (5-FU), 1 μg/ml cisplatin (CDDP), 100 pg/ml docetaxel (DOC), 50 μg/ml trifluorothymidine (TFT), 1 μg/ml cetuximab, 5 ng/ml bortezomib or 10 μg/ml zoledronic acid. Cells were also exposed to 15 Gy radiation in an X-ray irradiator (MBR-1505R2, 150 kV, 5 mA, filter: 1.0 mm aluminum, Hitachi Medico, Tokyo, Japan). After 48 h, 25 μl MTT was added to each well. After 4 h, dimethyl sulfoxide (100 μl/well) was added and the absorbance was measured with a spectrophotometer (BioRad Laboratories) at 490 nm. All assays were run in triplicate.

HUVEC were cultured in EGM-2 complete medium for one week and used for experiments (passages 5 to 7). Cells (1.5–2 × 10⁴ per well) were seeded into 96-well plates (Becton Dickinson, San Jose, CA, USA) and incubated for 24 h. Then, cells were incubated for 2 h with free CE diluted to the polyphenols concentrations of 2, 5, or 10 μg GAE/mL culture medium (M199 5% FBS). Also, HUVEC were incubated for 2 h with different types of empty or CE (2 μg/mL GAE)-loaded NPs based on Ch-der (QA-Ch or QA-Ch-S-pro) or PLGA NPs. The concentration of 2 μg/mL GAE was the maximum non-toxic load previously tested [20 (link),21 (link)]. The NPs were freshly prepared and diluted in culture medium to the desired concentrations. To study the anti-inflammatory effect of loaded NPs, DEXA (5 μg/mL culture medium) was used as positive control. After a 2-h incubation, cells were washed twice with PBS and treated with LPS (10 μg/mL) for 24 h to induce inflammatory stress. At the end of the treatments, the supernatants were collected and stored at −80° C for further analysis. Cell viability, before and after LPS treatment, was measured using the WST-1 assay, and the values obtained were compared with 100% of control (untreated cells). All treatments were done in triplicate and all experimental data resulted from at least two triplicate runs.

DESCs were seeded into 96-well plates (Becton Dickinson) at a cell density of 1 × 10³ cells/well for 24 h. Cells were treated with 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, and 50 ng/ml TNF-α and different concentrations of BMS-345541 including 0.01 μmol/L, 0.1 μmol/L, 1 μmol/L, 10 μmol/L, and 50 μmol/L. After treatment, the cells were enumerated using a Cell-Counting kit-8 (CCK-8, Dojindo, Tokyo, Japan) according to the manufacturer’s instructions every day. Briefly, 100 μl serum-free epithelial cell medium with 10 μl reagent was added to each well and incubated at 37 °C for 1 h. The reaction was measured at 450 nm using a spectrophotometer (Thermo Scientific Varioskan Flash, Thermo Scientific).

Cells were seeded in 96-well plates (Becton Dickinson, Franklin Lake, NJ, USA) at a density of 1 × 10³ cells/well and incubated for 24 h to allow for the attachment of cells. The cells were incubated with doxo, STDO-doxo and LSTDO-doxo at the same concentrations for 96 h. The cytotoxicity was correlated with the cell viability as evaluated by the CellTiter-Glo^® Luminescence Assay (Promega, Madison, WI, USA) with an Infinite200 PRO instrument (Tecan, Männedorf, Switzerland).