Imark microplate absorbance reader

A total of 2500 or 5000 cells/well were seeded in 96 well plates and were treated 24 h later with 0.1, 1, 10 and 100 µM of cisplatin (Sigma-Aldrich) or drug vehicle. After 72 h, 20 µL of Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) was added to each well, and absorbance was measured at 490 nm in iMark microplate absorbance reader (Bio-rad, Hercules, CA, USA). The IC50 of each RMS cell line was calculated with a nonlinear regression.
A total of 5000 cells/well of RH30 shControl and shHMGB1 were seeded in 96 well plates and treated 8 h later with 5, 10 and 20 µM of cisplatin (Sigma-Aldrich) or drug vehicle. After 48 h, 20 µL of Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) was added to each well, and absorbance was measured at 490 nm in iMark microplate absorbance reader (Bio-rad, Hercules, CA, USA).

Active FGF23 and DMP1 intracellular and extracellular levels were evaluated in cell lysates and culture media using mouse FGF23 ELISA kit and DMP1 ELISA Kit (Abcam, Cambridge, UK), respectively, according to the manufacturer’s instructions. MLO-Y4 cells were seeded in 12-well plates and treated with 17β–E2 or inhibitors for 4, 24 or 48 h, as described above. Cell lysates were performed detaching and collecting MLO-Y4 by centrifugation at 130× g for 10 min. Cells, washed in cold phosphate buffer saline (PBS), were suspended in PBS and sonicated four times. Then, cell lysates were centrifuged at 1500× g for 10 min at 4 °C and FGF23 and DMP1 assays were performed in the supernatants. Absorbance was measured at 450 nm with iMarkTM Microplate Absorbance Reader (BioRad, Hercules, CA, USA). Data were normalized on total protein content and FGF23 and DMP1 levels were expressed as a percentage of the respective levels measured in control set as 100%.

The cytotoxicity of all these EOs was evaluated in an HFF cell line, using a CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, United States), according to the manufacturer’s instructions. HFF cells (1 × 10⁵ cells/well) were cultured in 96-well plates at 37°C, in an atmosphere containing 5% CO₂, for 24 h. The cells were treated with varying concentrations of EOs or sulfamethoxazole (SMZ), and incubated for 24 h. Different concentrations of each EO were added to the wells. A 1.56% solution of DMSO in DMEM was used as the vehicle control. After incubating for 48 h, 20 μL of MTS solution, containing phenazine ethyl sulfate, was added to each well, and incubated for 3 h at 37°C. Absorbance was measured at 490 nm using an iMark^TM Microplate Absorbance Reader (BioRad, Hercules, CA, United States). Wells containing cells treated only with DMEM were used as the negative control (Montazeri et al., 2019 (link)). The 50% cytotoxic concentrations (CC₅₀) were calculated using Graph Pad Prism 8.0. The cytotoxicity experiment was performed in triplicate, using three separate plates.

hMSCs (1 × 10⁴ cells/samples) were seeded onto the samples and cultured for 6 h, 3 days, 7 days, and 9 days (cell proliferation), in DMEM. Quantitative analysis of cell proliferation on the samples was performed using a WST‐1 assay (Premix WST‐1 Cell Proliferation Assay System, Takara Bio Inc., Kusatsu, Japan). hMSCs (4 × 10⁴ cells/sample) were cultured for 14 days on samples in osteogenic differentiation medium. Alizarin Red S (Sigma‐Aldrich, USA) staining was used to confirm the osteogenic differentiation (according to the degree of mineralization) of hMSCs on sample surfaces. The stained cells were destained with cetylpyridinium chloride (Sigma‐Aldrich), and the extracted stains were measured using an absorbance reader (iMarkTM Microplate Absorbance Reader, Bio‐Rad, Hercules, CA, USA) at 595 nm to quantify the osteogenic differentiation of hMSCs.

HepG2 cells have low levels of cytochrome P450 enzymes compared with primary hepatocyte^{37 (link)} whereas the expression levels of BCRP in HepG2 cells is comparable to that in primary hepatocyte^{38 (link)}. Therefore, the inhibitory effects of BCRP-mediated efflux of MTX against cell variability and cytotoxicity was evaluated in HepG2 cells according to a previous report^{39 (link)}. Briefly, HepG2 cells were seeded on a 96 well-plate at a density of 2.5 × 10⁴ cells/well. After 24 h of seeding, HepG2 cells were incubated with the culture medium containing MTX in the presence or absence of 1 μM chrysin (a potent BCRP inhibitor)^{33 (link)} or 0.1 μM FBX. After 24 h of exposure, the cell viability in HepG2 cells was determined using CellTiter 96^® AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) following the instructions provided by the manufacturer. The absorbance was measured at 490 and 620 nm with the iMark^TM Microplate Absorbance Reader (Bio-Rad, Hercules, CA). The cytotoxicity in HepG2 cells was determined using CytoTox-Glo™ Cytotoxicity Assay (Promega, Madison, WI) following the instructions provided by the manufacturer. The luminescence was measured with 2300 Multiplate Reader ARVO^TM X2 (PerkinElmer, Waltham, MA). The cell viability and cytotoxicity in HepG2 cells after treatment of vehicle (control) were set at 100%.

The HCT116 colon cancer cells or ASCs were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and cultured for 12 h. TEx, AEx, ^99mTc-TEx-Cy7 and ^99mTc-AEx-Cy7 at various concentrations (i.e., 0, 5, 10, 50 and 200 μg/mL) were added to the medium, and then the cells were incubated for another 24 h. In addition, TEx (200 μg/mL), AEx (200 μg/mL), ^99mTc-TEx-Cy7 (37 KBq) and ^99mTc-AEx-Cy7 (37 KBq) were incubated for different time points (0, 2, 6, 12,18, 24, 48 and 72 h). At the end of the incubation, CCK-8 was added and continued to incubate for 2 h. The absorbance values of the cells were measured with a microplate reader (iMarkTM Microplate Absorbance Reader, Bio-Rad, USA) at 450 nm.