Cell proliferation elisa brdu assay kit

Cells were incubated with 2.5 µM of PdNPs, 0.75 mM of MLT, 2.5 µM of PdNPs combined with 0.75 mM of MLT, or 5 µM of DOX for 24 h. The 5-bromo-2′-deoxyuridine (BrdU) labeling solution was added to the culture medium 2 h before the incubation period ended. Cells were fixed and the level of incorporated BrdU was determined using the Cell Proliferation ELISA BrdU assay kit (Roche), following the manufacturer’s instructions. The proliferation activity of untreated cells at 0 h was considered to be 100%.

Recombinant VEGF-A was from PeproTech (Rocky Hill, NJ, USA). DMEM, fetal bovine serum (FBS), TrypLE™, Medium 199 (M199), and all cell culture reagents were from Invitrogen (Carlsbad, CA, USA). Antibodies against VEGFR-2, anti-phospho-VEGFR-2 (Y1175), Akt and anti-phospho-Akt (S473), ERK1/2 and anti-phospho-ERK1/2 (T202/Y204), FAK, anti-phospho-FAK (Y397), Src and anti-phospho-Src phosphorylated (Y416), were from Cell Signaling (Danvers, MA, USA). Antibody against α-tubulin and anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase antibodies were from GeneTex Inc (Irvine, CA, USA). All materials for immunoblotting were from Bio-Rad (Hercules, CA, USA). BD Matrigel^TM basement membrane matrix was from Becton Dickinson (Mountain View, CA, USA). The immobilon Western chemiluminescence HRP substrate was from Millipore (Billerica, MA, USA). Cell Proliferation ELISA, BrdU assay kit was from Roche (Indianapolis, IN, USA). Toluidine blue O, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA).

Cell proliferation was determined according to method described earlier [85 (link)] and also manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA). Cells were incubated with various concentrations of GO, AgNPs or GO-AgNPs for 24 h; the BrdU labeling solution was added to the culture medium 2 h before the end of the incubation. Cells were fixed and the level of incorporated BrdU was determined using the Cell Proliferation ELISA BrdU assay kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. Proliferation activity of the untreated cells at the time point of 0 h was considered as 100%.

BrdU incorporation was performed utilizing Cell Proliferation ELISA, BrdU Assay Kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s instruction. In brief, 4x10³ cells (in 100 μl/well) were cultured in 96-well plates in complete growth media. At indicated time points cells were labelled using 10 μM BrdU and re-incubated overnight at 37°C in a humidified atmosphere. The next day, the culture media was removed; cells were then treated with FixDenat and subsequently incubated with the anti-BrdU-P OD antibody for 90 minutes at room temperature. After the incubation period cells were washed and supplemented with the substrate solution. Accordingly quantification of the reaction product was measured by using a scanning multi-well spectrophotometer (ELISA reader) at an absorbance of 370 nm with a reference wavelength of 492 nm. For comparison, we show for indicated time points the percentage of BrdU-positive cells in control (untreated) or DMSO (solvent control) or resveratrol treated cells.

Recombinant human LIGHT (rhLIGHT) was purchased from R&D Systems (Minneapolis, MN), and diluted in 0.1% BSA-PBS buffer. The CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Madison, WI, USA). StemPro^® MSC SFM CTS^™, StemPro^® Adipogenesis Differentiation Kit, StemPro^® Chondrogenesis Differentiation Kit, StemPro^® Osteogenesis Differentiation Kit, and fetal bovine serum (FBS) were obtained from GibcoBRL (Grand Island, NY, USA). Oil Red O staining kit (for adipocytes), Alcian blue staining kit (for chondrocytes) and Alizarin red staining (for osteocytes) were purchased from Invitrogen (Camarillo, CA, USA). The antibodies for western blotting, namely, anti-CDK2, cyclin E, and β-actin, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p27, p-STAT3, p-Smad3, STAT3, Smad3, CDK1, cyclin B1, cyclin D1, and cyclin D3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-HVEM, LTβR, CD19, CD34, CD45, CD44, CD90, and CD105 antibodies and PI/RNase solution were purchased from BD Bioscience (San Jose, CA, USA). ELISA for PDGF-BB and TGF-β1 were purchased from R&D Systems. The Cell Proliferation ELISA, BrdU Assay Kit was purchased from Roche Diagnostics (San Francisco, CA, USA). All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).