Anti brdu primary antibody

For BrdU incorporation analysis, each sample at the last half hour of time point was pulse 30 min with 10 µM of BrdU and after each time point, cells were fixed and permeabilized with 70% ethanol and the histones were dissociated with 2 M HCl to allow the antibody entry. Acid pH were neutralized by Na₂B₄O₇ 0.1 M pH 8. After extensive washes with PBS/0.5% BSA/0.1% Triton, BrdU positive cells were detected with an anti-BrdU primary antibody diluted 1:100 (DAKO, SA, Glostrup, Denmark) and with an anti-mouse-Alexa 488 conjugated diluted 1:100 (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA). Both antibodies were incubated for 1 h RT in the dark. All samples were counterstained with propidium iodide for DNA content/BrdU biparametric analysis. BrdU percentage was calculated using the CytExpert v2.2 software (Beckman Coulter). Each analysis was performed by acquiring 10,000 events/sample.

Bromodeoxyuridine (BrdU) was added to the medium in the last 30 min of growth, and the cells were then fixed and permeabilized. Histones were dissociated with 2 M HCl as previously described [21 ]. BrdU-positive cells were detected with anti-BrdU primary antibody diluted 1:100 (DAKO; Santa Clara, CA, USA) and Alexa488-conjugated anti-mouse antibody diluted 1:100 (Thermo Fisher Scientific; Waltham, MA, USA). Both antibodies were incubated with the cells for 1 h at room temperature in the dark. BrdU fluorescence was measured using a CytoFlex flow cytometer, and S-phase analysis was performed with CytExpert v 2.3 software (Beckman Coulter, Brea, CA, USA). All samples were counterstained with propidium iodide (PI) for DNA/BrdU bi-parametric analysis.

Bromodeoxyuridine (BrdU) was added to the medium in the last 30 min of growth, and the cells were then fixed and permeabilized. Histones were dissociated with 2 M HCl as previously described (30 (link)). BrdU-positive cells were detected with anti-BrdU primary antibody diluted 1:100 (DAKO; Santa Clara, CA, USA) and Alexa488-conjugated anti-mouse antibody diluted 1:100 (Thermo Fisher Scientific; Waltham, MA, USA). Both antibodies were incubated with the cells for 1 h at room temperature in the dark. BrdU fluorescence was measured using a CytoFlex flow cytometer, and S-phase analysis was performed with CytExpert v 2.3 software (Beckman Coulter, Brea, CA, USA). All samples were counterstained with propidium iodide (PI) for DNA/BrdU bi-parametric analysis.