Xtt cell proliferation kit

Two viability tests with the use of the XTT cell proliferation kit ((2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide, Roche) and MTT cell proliferation assay (Thiazolyl Blue Tetrazolium Bromide, Sigma Aldrich) were conducted. According to ISO 10993-5 standard [22 ], the extracts were prepared in duplicates of each variant of material by incubation in a supplemented DMEM medium w/o Phenol Red (1 mL per 3 cm² of disc surface, according to ISO 10993-12) [23 ] for 24 h (37 °C, 5% CO₂). After that time, extracts were added to cells and they were incubated for the next 24 h. As a negative and positive control (NC and PC, respectively), DMEM medium w/o Phenol Red (NC) and DMEM medium w/o Phenol Red with 0.1% (v/v) Triton X (Sigma Aldrich) (PC) were used. In the next step, extracts were removed and XTT or MTT solution (1 mg/mL in DMEM without supplements and Phenol Red) was added. After 4 h of incubation, in the case of the XTT test, the absorbance of resulting solutions was measured with the use of a UV–Vis plate reader (Epoch, BioTek, Winooski, VT, USA, Gen5 software) at 470 nm with a reference wavelength at 650 nm. In the case of the MTT test, the solutions were removed from wells and the crystals were dissolved in 100 µL of isopropanol. The absorbance was measured at 570 nm, reference wavelength 650 nm.

Cell proliferation was detected using an XTT cell proliferation kit (Roche, Basel, Switzerland). In short, cells (1 × 10³) were seeded in a 96-well plate and cultured with different treatments. A mixture of XTT reagents, which includes PBS, XTT labeling reagents, and electron coupling reagents, was added to each well and cultured with cells at 37 °C for 4 h. Finally, the absorbance at 492 nm was read, and relative cell viability was expressed as a fold change over the mean of the first day. Cell migration and invasion abilities were determined using Transwell cell migration assay kits (Corning, New York, NY, USA) and BioCoat™ Matrigel^® Invasion Chambers (Corning), respectively. Briefly, cell samples (1 × 10⁵ cells) were suspended in serum-free medium and seeded in 24-well transwell chambers. Different media containing 1% FBS were added to the lower chamber. After incubation, the migrated/invaded cells were stained and counted using microscopy.

Hematopoietic and non-hematopoietic cell lines were reciprocally cultured in RPMI 1640 and DMEM (Pan Biotech), supplemented with 20% fetal bovine serum (Pan Biotech). Cell viability was analyzed using the XTT Cell Proliferation Kit (Roche Applied Science), four days after treatment with PyQ. CD45 shRNA lentiviral particles (sc-29251-V) and control particles (sc-108080) (Santa Cruz Biotechnology, Inc.) were used to knock down the CD45 on THP1.

Primary human osteoblasts of the first passage were plated out in a 96 wells plate at a density of 4.000 cells/well. After 24 hours cells were exposed to medium with 25(OH)D₃ (0, 100, 200 or 400 nmol/L) or 1,25(OH)₂D₃ (0, 1, 10 or 100 nmol/L). Medium was replaced every 3 days by complete medium with or without 25(OH)D₃ or 1,25(OH)₂D₃. The proliferation of primary human osteoblasts was measured at day 3 and 6 using the XTT Cell Proliferation Kit (Roche Diagnostics) according to the manufacturer’s protocol. Briefly, cells were incubated with the XTT solution at 37°C, whereby the viable cells formed an orange formazan dye by cleaving the yellow tetrazolium salt XTT. After 2 hours the orange formazan solution was quantified by a photospectrometer (Berthold Technologies) at 450 nm.