J. Biol. Chem., Vol. 282, Issue 1, 287-293
Cell Proliferation—INS832/13 cell proliferation was evaluated using a BrdUrd3 enzyme-linked immunoassay kit (Roche Applied Science). Adenovirus-transduced INS832/13 cells were seeded in 96-well plates (8 x 104 cells/well) and allowed to recover for 24 h. BrdUrd was added to the culture medium for 1 h, and cells were fixed, incubated with peroxidase-conjugated anti-BrdUrd antibody, and the immune complexes were quantified by enzyme-linked immunosorbent assay (Bio-Rad).
J. Clin. Invest. 118(1): 40-50 (2007)
In vivo proliferation assay. Pregnant females were injected intraperitoneally with 100 μg/g body weight BrdU (Sigma-Aldrich). After 1 hour, mice were euthanized and embryos recovered for immunohistochemistry as described in Supplemental Methods. BrdU-positive cells were detected with the BrdU staining kit (Zymed) followed by staining in Mayer hematoxylin. Percent proliferative cells was determined by blinded quantitation of BrdU-positive cells in the lymphatic or vascular endothelium divided by total hematoxylin-stained nuclei. Data were analyzed using the 2-tailed Student’s t test assuming unequal variance. A P value < 0.05 was considered significant.
J. Clin. Invest. 118(1): 89-99 (2007)
Endothelial cell isolation and proliferation assay. Primary endothelial cells were isolated from adult mouse livers following mechanical mincing and collagenase (2 mg/ml) digestion for 30 min at 37°C. Cells were isolated by incubating for 30 min at 23°C with magnetic Dynabeads (Invitrogen) labeled with anti-CD31 antibodies and using a magnetic separator (53). Cells were then incubated in DMEM supplemented with 20% FBS, penicillin/streptomycin (100 U/ml), heparin (100 μg/ml), endothelial cell growth factor (100 μg/ml), 1× nonessential amino acids, 2 mM l-glutamine, 1× sodium pyruvate, gentamicin (58 μg/ml), and amphotericin B (25 ng/ml), termed “complete endothelium medium.” To assess the effects of VEGF-A on endothelial cell proliferation, cells from passage 1 or 2 were plated for 4 h in 96-well cell culture plates coated with fibronectin (5 μg/ml) at a density of 4,000 cells per well. The medium was then replaced with fresh complete endothelium medium containing 2% FBS and 0.5% BSA. Cells were incubated at 37°C for 48 h in the absence or presence of 50 ng/ml VEGF-A, prior to the addition of One Solution reagent (Promega) for 2 h. Optical density was then determined at 490 nm on a Molecular Devices microplate reader.
J. Clin. Invest. 118(1): 217-228 (2007).
Cell counting and TB exclusion assays. 7.0 × 104 cells were seeded in 60-mm plates 1 day prior to initiation of experiment. Starting at day 0, cells were trypsinized and stained with 0.4% TB solution (Sigma-Aldrich), and both viable and dead cells were counted in a Bürker chamber. Every other day, starting at day 0, cultures for subsequent counting were supplemented with fresh media containing 1% FCS, PEST, and DMSO or DAPT. Each experiment was performed in triplicate and repeated twice.
