Rat GEnCs were kindly provided by Professor F. R. Danesh (MD Anderson cancer center, Houston, TX) and maintained at 37 °C in a humidified atmosphere containing 5% CO2, cells were cultured in RPMI 1640 Medium (Gibco by Invitrogen, Carlsbad, CA) containing 5.5 mM D-glucose (Sigma-Aldrich, St. Louis, MO), 10% fetal bovine serum (Gibco by Invitrogen), and 10% Nu-Serum I Culture Supplement (BD Biosciences, San Jose, CA). At 70% confluence, GEnCs were treated with or without 30 mM D-glucose in the presence or absence of a selective ROCK inhibitor, Y27632 (10 μM; Sigma-Aldrich). Mannitol (24.5 mM) was used as an osmotic control. To overexpress ROCK1, a ROCK1 adenovirus (Ad-ROCK1, Vectorbiolabs, Philadelphia, PA) was added to the medium bathing GEnCs at 1:2,000 dilutions for 2 days. β-galactosidase adenovirus (Ad-β-Gal, Vectorbiolabs, Philadelphia, PA) was used as a control for virus infection.
Ad β gal
Manufactured by Vector Biolabs
Ad-β-Gal is an adenoviral vector that expresses the E. coli β-galactosidase (lacZ) gene. It can be used to monitor adenoviral transduction efficiency in target cells.
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Lab products found in correlation
2 protocols using ad β gal
1
Rat Glomerular Endothelial Cell Culture
2
Dominant-Negative Rac1 Inhibits Cardiomyocyte Migration
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Wild‐type neonatal cardiomyocytes were prepared and cultured, as described previously.21 (link) Cardiomyocytes were infected with adenoviruses expressing a dominant‐negative form of Rac1 (Ad‐Rac1N17; Vector Biolabs) or β‐galactosidase (Ad‐β‐gal; Vector Biolabs) as a control at a multiplicity of infection of 10 plaque forming units per cell. The scratch was performed 6 hours after adenoviral infection. Cells were fixed and immunostained with primary antibodies for Rac1 (Santa Cruz Biotechnologies) and α‐actinin (Sigma‐Aldrich) 24 hours after scratch.
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