The SEA was prepared at Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA). The amount of gram negative bacterial endotoxin present in the SEA preparation was quantified using the endpoint chromogenic limulus amebocyte lysate assay (QCL1000, Lonza). Mouse cholangiocyte line 603B, primary human cholangiocytes (from Life Technologies, Carlsbad, CA, USA), normal human cholangiocyte cell line H69 (gift from Professor G.J. Gores (Mayo Clinic) and Professor G.D. Alpini (Texas A&M University System Health Science Center)) and clonally derived rat hepatic stellate cell line 8B (gift from Professor M. Rojkind (George Washington University, in memoriam)) were incubated with 10g μg/mL of SEA or 0.0001μg/mL LPS (control, same amount of endotoxin present in the SEA preparation) for 2,6,12 and 24 hours. RNA and conditioned media were collected for analysis. Cell proliferation was assessed by BrdU incorporation (6813, Cell Signaling Technology) after 24h of SEA or control incubation.
Brdu incorporation
Manufactured by Cell Signaling Technology
BrdU incorporation is a laboratory technique that measures the incorporation of the synthetic thymidine analog bromodeoxyuridine (BrdU) into newly synthesized DNA during cell division. This method provides a quantitative assessment of cell proliferation.
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2 protocols using brdu incorporation
1
Cholangiocyte and Hepatic Stellate Cell Response to Bacterial Endotoxin
2
BrdU-Based Cell Proliferation Assay
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DNA synthesis in proliferating cells was detected by BrdU incorporation (Cell Signaling Technology, Danvers, MA). Parental and ARNT knockdown cells were spread onto 96-well plates and incubated for 24 h. 5-bromodeoxyuridine (BrdU) reagent was added to culture media for 0 to 48 h, 100 µl Fixing Solution was added to the cells for 30 min. The cells were washed with Wash Buffer and incubated for 1 h with 100 µl 1 x BrdU antibody. After adding 100 µl 1 x HRP-conjugate solution for 30 min, 100 µl TMB substrate solution was added. Following 30 min incubation, the stop solution was added. The OD was measured at 450 nm using a plate reader. For immunofluorescence, cells transfected with 30 nM ARNT siRNA oligonucleotides were pulse labeled with 20 nM BrdU for 20 min or 24 h. Cells were fixed and stained with anti-BrdU antibodies followed by anti-mouse FITC and DAPI. The percentage of cancer cells with positive nuclear and BrdU staining was calculated by counting immunopositive cells in four randomly chosen using Image J software.
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