Brdu cell proliferation kit

A BrdU cell proliferation kit from Cell Signalling Technology was used to evaluate effect of NOSTRIN on HCT116 cell proliferation as described previously [22 (link)]. Stable HCT116 cells over-expressing either the control vector or the Nostrin cDNA were seeded (1 X10⁴ cells/well) in triplicates in 96-well plates and cultured for 24 h in the presence of BrdU solution. Cells were then incubated with a fixing/denaturing solution after removing the media, at room temperature for 30 min, followed by incubation of the cells with detection antibody for 1 h. The cells were washed and incubated with HRP-conjugated secondary antibody solution for 30 min. Following washing cells were incubated with tetramethylbenzidine (TMB) substrate, and finally the reaction was stopped after 15 min with a STOP solution. Colorimetric quantification was done by measuring the absorbance at 450 nm wavelength using a multimode plate reader (PerkinElmer, USA).

Cell Proliferation was also measured by the incorporation of BrdU in cells by using the BrdU cell proliferation kit from Cell Signaling. MDA-MB-231 cells were plated in 96-well plates and treated with 30 uM curcumin and/or 1 uM ATRA for 48 hours. Controls were treated with 0.1% DMSO, 0.1% ethanol and/or the combination of both. Cells were then pulsed with BrdU overnight, fixed and followed by immunodetection of the incorporation of BrdU label. Absorbance was read at 450 nm to determine cell proliferation.

Cellular proliferation rate was evaluated by using BrdU cell proliferation kit (Cell Signaling, Beverly, MA) conducted as previously described.^[5b]

Cell proliferation of AtT-20 cells in hypoxic conditions was measured by a BrdU incorporation assay. Cells under different treatments were plated onto 96-well plates. These cells were subjected to BrdU using the BrdU Cell Proliferation Kit (Cell Signaling, USA) following the manufacturer’s instructions. Three independent experiments were performed. Next, we employed Multisizer 3 COULTER COUNTER to count the AtT20 cell numbers. Before counting the sample, flush the system and count PBS as a blank control. Put 0.2 ml of suspension of cells into 10 ml PBS, and then mix the sample thoroughly. Turn the glass valve directly above the cells to vertical, and then turn it back to horizontal to begin the count. The count displayed is the the number of cells in 0.5 ml.

The antibodies were purchased as follows: Rabbit polyclonal anti-JNK-1 (S-18), anti-ERK-1 (K-23) and anti-p38 (N-20) antibodies were all purchased from Santa Cruz (Dallas, TX, USA). Anti-phospho-ERK (T202/Y204) and anti-phospho-JNK (T183/Y185) were purchased from Cell Signaling Technology (Hitchin, UK). Anti-phospho-p38 (44684-G) was purchased from Applied Biosystems, Life Technologies (Paisley, UK). HRP- conjugated anti-mouse, and conjugated anti-rabbit antibodies were purchased from Jackson Immunoresearch laboratories (Luton, Beds, UK). Anti-Mouse CD34-FITC conjugated, anti-Mouse CD115 (c-fms)-PE conjugated, and recombinant M-CSF were all purchased from eBioscience (Hatfield, UK). SP600125 JNK inhibitor, SB 203580 p38 inhibitor, and U2106 MEK1,2 inhibitor was purchased from Tocris Bioscience (Bristol, UK). BrdU Cell Proliferation kit was purchased from Cell Signaling (New England Biolabs, Hitchin, UK). SuperScript^® III First-Strand Kit (cDNA synthesis system for RT-PCR), oligonucleotide PCR Primers, SYBR^® Select real-time PCR Master Mix were purchased from Applied Biosystems, Life Technologies (Paisley, UK). RNeasy Mini Kit (RNA extraction kit), and RNase-Free DNase Set were obtained from QIAGEN (Manchester, UK). All other materials used were of the highest commercial grade available and they were obtained from Sigma Aldrich (Poole, Dorset, UK).