5 bromo 2 deoxyuridine brdu cell proliferation assay kit

Cell growth curves were assessed by using the Cell Counting Kit-8 (CCK-8) (Abmole, Houston, TX, USA). Cells were seeded at a density of 2000 cells per well in 96-well plates and incubated for 1, 2, and 3 days, respectively. Then, the cells were incubated with CCK-8 solution for 2 h. The optical density (OD) values were measured on the microplate reader (Infinite M200 Pro, Tecan Group, Männedorf, Switzerland) at a wavelength of 450 nm. Similarly, cell proliferation was quantified on day 2 using a 5-bromo-2-deoxyuridine (Brdu) Cell Proliferation Assay Kit (BioVision, Milpitas, CA, USA).

Collagenase was obtained from Worthington Biochemical Corporation (Labclinics, Madrid, Spain). Cell Lytic for cell lysis and protein solubilization, crystal violet, protease inhibitor cocktail (Complete, EDTA-free), thapsigargin and Tween-20 were purchased from Sigma Chemicals Co. (Madrid, Spain). Fetal bovine serum, Hank’s balanced salts (HBSS), horse serum, medium 199 and SuperSignal West Femto were obtained from Fisher Scientific Inc. (Madrid, Spain). Polystyrene plates for cell culture were obtained from Thermo Fisher Sci. (Madrid, Spain). Penicillin/streptomycin was purchased from BioWhittaker (Lonza, Basel, Switzerland). Acrylamide, Bradford´s reagent, Tris/glycine/SDS buffer (10×) and Tris/glycine buffer (10×) were from Bio-Rad (Madrid, Spain). 5-Bromo-2-deoxyuridine (BrdU) cell proliferation assay kit was purchased from BioVision (Deltaclon S.L., Madrid, Spain). SB203580 and U0126 were obtained from Tocris (Biogen Científica, Madrid, Spain). Species-specific HRP-conjugated secondary antibodies were purchased from Thermo Fisher Sci. (Madrid, Spain). All other analytical-grade chemicals used were obtained from Sigma Chemicals Co. (Madrid, Spain).