5 bromo 2 deoxyuridine brdu cell proliferation assay kit

HSV SMCs and ECs were seeded in clear, 96-well plates at a density of 5 × 10³ cells/well and quiesced in full medium, without serum for 48 or 24 h, respectively. The cells were then stimulated with a range of concentrations of ligands ±5% FCS, over a period of 48 h (SMCs) or 24 h (ECs). Cell proliferation was assessed using either Cell Titer 96 aqueous nonradioactive cell proliferation assay (MTS; Promega, Madison, Wis., USA) or the 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling Technology, UK) according to manufacturer's instructions. Colorimetric output was measured on a Wallac VICTOR² plate reader at absorbance values of 490 or 450 nm for the MTS and BrdU assays, respectively.

Myoblast proliferation was assessed using the Celltiter 96 aqueous one solution cell proliferation assay kit (Promega), which is a non-radioactive colorimetric method for determining the number of viable cells in culture. The assay is composed of a tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-H-tetrazolium, inner salt, MTS) and an electron-coupling reagent (phenazine methosulfate (PMS)). Myoblasts were trypsinized and counted, before seeding at a concentration of approximately 1000 cells per well in 96-well plates (final volume: 100μL), and incubated at 37°C, 5% CO₂ for 24h. Treatments were added to the culture media and the cells were further incubated at 37°C, 5% CO₂ for 48h. The proliferation assay was subsequently performed according to the manufacturer’s protocol. Incubation with IGF1, a known activator of myoblast proliferation (Milasincic et al. 1996 (link)), was used as a positive control in these experiments. Selected results generated using the above methodology were validated using a 5-bromo-2′-deoxyuridine (BrdU) Cell Proliferation Assay Kit (Cell Signaling). The assay was performed according to the manufacturer’s protocol using similar seeding densities and conditions described above.

For the in vitro immune suppression assay, murine splenocytes were used as responder cells and phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) as a mitogen. Splenocytes (2 x 10⁶ cells) were first pre-stimulated with 200 ng/ml of PMA and then co-cultured for 24 h by adding to TMSC (3 x 10⁴ cells) pre-adhered in a 96-well plate (BD Bioscience). After co-culture, the proliferation of splenocytes was measured using a 5-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit (Cell Signaling Technology, Boston, MA, USA) as previously described [28 (link)]. Briefly, splenocytes were incubated with exogenous BrdU for 3 h and centrifuged. The cells were then fixed with fixing solution for 30 min. The incorporated BrdU was probed using an anti-BrdU antibody, followed by the corresponding HRP-conjugated secondary antibody solution. Cells were washed, and absorbance was measured at 450 nm.