Brdu proliferation assay kit

The effect of mitomycin C on the proliferation of NBL-6 was evaluated by using a bromodeoxyuridine (BrdU) proliferation assay kit (Abcam, Cambridge, MA, USA). To this end, NBL-6 were seeded on a 96-well plate at a density of 5 × 10⁴ cells per well and incubated with mitomycin C for 48 hours. Regular medium was used to establish baseline proliferation. The BrdU proliferation assay kit was used in accordance with the instructions of the manufacturer, and the resulting absorbance was read at 450 nm on a Multiskan EX microplate reader by using Ascent software (Thermo Scientific, Waltham, MA, USA). Empty wells and wells without BrdU were included as controls.

Cells (5000 per well) were plated to each well in a 96-well plate. After 72 h drug treatment, cell proliferation activities were evaluated by BrdU proliferation assay kit (Abcam, Cambridge, UK). For combination studies, the IC₅₀ of each drug was first determined. The cells were then treated with increasing doses of single drug alone or an equipotent constant-ratio combination of both drugs. After 72 h drug treatment, proliferation activity was determined. The CI was calculated using the CalcuSyn software (Biosoft, St. Louis, MO). If CI is less than 1, the combination is synergistic; if CI is equal to 1, the combination is additive; if CI is more than 1, the combination is antagonistic (Huang et al. 2017 (link)).

Cells were plated in 96-well plate in culturing medium and treated with narasin for 72 h. Cell proliferation was assessed using BrdU proliferation assay kit (Abcam). 20 µl of BrdU working solution was added into each well and incubated at 37 °C for 3 h. The spectrometric absorbance was measured at 490 nm.