5 bromo 2 deoxyuridine brdu

For apoptosis, cells were seeded and treated with PA-2 for 24 h, trypsinized and stained with Annexin V-FITC (100X dilution; Invitrogen, Carlsbad, CA) and PI (0.5 μg/ml; Sigma, St Louis, MO), then analyzed by FACScaliber (BD Biosciences, San Jose, CA). To determine cell proliferation, we measured the incorporation of 5-bromo-2′-deoxyuridine (BrdU) into newly synthesized cellular DNA followed by the manufacture’s protocol (BD Biosciences), and cells were subjected to flow cytometric analysis. Cell cycle phase distribution was analyzed by flow cytometry as described [15 (link)].

A DNA synthesis–based cell proliferation assay using 10 mM 5-Bromo-2´-Deoxyuridine (BrdU) (BD Biosciences, Franklin Lakes, NJ, USA) was performed according manufacturer instructions.

All surgeries were performed according to the National Institutes of Health guidelines for the humane treatment of laboratory animals according to the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86–23) and with approval of the Institutional Animal Care and Use Committee of Vanderbilt University Medical School. Prior to hepatectomy or for sham operations, mice were anesthetized with 60 mg/kg ketamine (Hospira) and 7 mg/kg xylazine (Phoenix Pharmaceutics) and positioned supine. A transverse incision was made inferior to the xiphoid process, which was excised. The median and left lateral lobes were eviscerated and ligated resulting in 60% liver removal. About 5 mg/kg/day of recombinant human IGF-1 (Cell Sciences) dissolved in water was given continuously with an osmotic pump (DURECT). To quantitate dividing hepatocytes, mice received 1 mg intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; BD Pharmingen) 2 h before sacrifice. At tissue recovery, mice were anesthetized and weighed. Livers were excised, rinsed, blotted, and weighed. Sections were fixed in 10% neutral buffered formalin. Mortality after hepatectomy was < 5% and not associated with a particular genotype.

Cells were pulse-labeled with 10 μM 5-bromo-2-deoxyuridine (BrdU, BD Pharmingen, San Diego, CA) for 90min (37°C), washed in PBS, then fixed in 70% ethanol (1-3d, −20°C). DNA was denatured (2 M HCl, 30min, room temperature) and neutralized (0.1 M Na₂B₄O₇, 10min, room temperature). Cells were stained with APC-conjugated anti-BrdU antibody (BD Pharmingen) and 7-AAD (BioLegend, San Diego, CA), and analyzed by flow cytometry [54 (link)].