Chorley, B.N., Li, Y., Fang, S., Park, J-A. and Adler, K.B.
Notes: NHBE cells were seeded and grown in 12-well plates in growth medium until 60–79% confluency. Medium was replaced with medium free of antibiotics or serum to induce quiescence. Transfection reagent and DNA were prepared as follows: 2µl of FuGENE® 6 reagent was added to 48µl of serum- and antibiotic-free medium and incubated at room temperature for 5 minutes. Next 0.1nmol of 21-bp iNOS siRNA was added and incubated for 15 minutes. 50µl of the transfection complex was added to each well for a final concentration of 0.45% FuGENE® 6 reagent and 225nM iNOS siRNA. Cells were incubated at 37°C for 24 hours.
To determine what, if any, protein kinases were involved in (R)-albuterol upregulation of iNOS message, inhibitors of several protein kinases were used to treat the NHBE cells. Because, protein kinase C inhibitors appeared to attenuated the (R)-albuterol-mediated iNOS expression, the PepTag® Non-Radioactive PKC Assay was used to measure protein kinase C activity in response to (R)-albuterol treatment.
All reagents were tested for cytotoxicity to NHBE cells using an LDH release assay. (4273)