Brdu elisa kit

Cell viability was determined using a MTT metabolism test as described [44 (link)]. Briefly, at given times after treatment, MTT stock solution was added to a final concentration of 0.5 mg/mL and after 1 h formazan crystals were dissolved in DMSO. Optical densities were measured at 570 nm and 620 nm using a scanning multiwell spectrophotometer. Cell proliferation was assessed using ELISA BrdU kit (Roche Diagnostics GmbH, Germany) according to the manufacturer’s protocol. For MTT and BrdU assays, cells were seeded at a density of 4 × 10³ cells/well.

The proliferation of 5637, T24 cells and T24 glycoengineered cell models was evaluated in basal and silenced-CD44 conditions, using the cell proliferation ELISA BrdU Kit (Roche Diagnostics GmbH), according to the manufacturer's instructions. The immunoassay results were monitored at 450 nm using the iMARK™ microplate reader (Bio-Rad). Cell death negative controls composed of 1% Triton-X in a complete cell culture medium were used. The results are presented as the average and standard deviation of three independent assays with three replicates each.

A ready-for-use ELISA BrdU Kit from Roche Diagnostics (Mannheim, Germany) was used to assess cell proliferation. The test was carried out in accordance with the manufacturer’s instructions provided. Initially, appropriate cell densities (as above) of melanoma and normal cells were plated on microtiter plates (NEST). After 24 h, the cells were exposed to increasing concentrations of DAP (2–200 µM). The next steps of the BrdU assay have been previously described [19 (link),20 (link)].

FLS were isolated from WT or Batf knockout (KO) mice and cultured as described by Zhao et al. [23 (link)]. FLS of passages 4–8 were used for further analysis. Pure FLS (> 90% CD90⁺/< 1% CD14⁺) were identified by flow cytometry using antibodies against CD90 and CD14 (Abcam). FLS proliferation in culture was quantified by measuring bromodeoxyuridine (BrdU) incorporation [20 (link)]. Briefly, FLS cultured in a 96-well plate were treated with or without tumor necrosis factor (TNF)-α (100 ng/ml), and BrdU labeling was detected using the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit (Roche). Proliferating cells in synovial sections were identified by detecting Ki67 using an antibody obtained from Abcam. The empty adenovirus (Ad-C) and BATF-expressing adenovirus (Ad-Batf) were as previously described [4 (link)]. FLS were infected with Ad-Batf and Ad-C at the indicated multiplicities of infection (MOI) for 2 h, washed, and maintained for 48 h before analysis.

DNA synthesis was measured by the cell proliferation ELISA BrdU kit (Roche Diagnostics, Basel, Switzerland). NIH3T3 cells were starved for 16 h. Cells were stimulated with either WT FGF2 or mutants on 96-well plate for 24 h and concomitantly BrdU solution was added to the culture. We also tested the mixture of WT FGF2 (5 ng/ml) and each mutant (250 ng/m). The amplitude of absorbance at 450 nm is proportional to the BrdU incorporation into the cells.