ROS measurements Flow cytometry with carboxy H2 DCFDA detection identified intracellular H2O2, which was used as a surrogate marker for superoxide generation. Oxidation of this non fluorescent substrate generates a green fluor escent product. Because the cell membranes are perme able to the selleck chem Pacritinib esterified form of carboxy H2 DCFDA, cells uptake it freely. The dye becomes trapped in the cells as a result of deacetylation by intracellular esterases and thus becomes available to oxidation by intracellular H2O2. Since superoxide is relatively short lived because it is rapidly dismutated to H2O2, intracellular H2O2 levels are therefore a more reliable indicator of intracel lular ROS burden. For the flow cytometry assay, cells were trypsinized using 0. 25% trypsin EDTA solution and resuspended in growth medium.
Cells were delivered into 5 mL polystyrene tubes, pelleted, and then incubated for 25 minutes at 37 C with carboxy H2 DCFDA mixed isomers reagent diluted to 2 uM in PBS supplemented with 0. 5% FBS. Cells were then pelleted again and incubated with growth media for 10 minutes at 37 C and washed Inhibitors,Modulators,Libraries twice with PBS FBS. In the final step, cells were pelleted, resuspended in propidium iodide solution for 10 min utes, and carboxy H2 DCFDA fluorescence emitted by 10,000 live cells was quantified using BD FACScan flow cytometer. Reverse Transcription PCR and Western blotting Total RNA was collected from N27 cells following the Trizol protocol. Messenger RNA was reverse transcribed using random primers to cDNA with Superscript II.
PCR was performed using NADPH Inhibitors,Modulators,Libraries oxidase subunit gene specific primers that yielded PCR products ranging between 400 and 500 bp. The following primer sets were designed based on the following accession numbers PCR products were separated by electrophoresis on 1% agarose gels and Inhibitors,Modulators,Libraries visualized with ethidium bromide. Specificity of each primer set was confirmed by amplification of a single band of expected size. For Inhibitors,Modulators,Libraries Western immunoblots, whole cell lysates were made in cell lysis buffer supplemented with 1% SDS, sonicated and separated on precast 4 12% SDS PAGE gels. Proteins were then transferred to PVDF membranes and blocked with 5% milk in TBS with 1% Tween 20. The membranes were then incubated overnight at 4 C with the following primary antibodies anti p47phox, anti Nox2, and anti Inhibitors,Modulators,Libraries p22phox and anti p67phox. Membranes were then rinsed, incubated in alka line phosphatase conjugated secondary antibodies, and a chemiluminescent signal was detected using Lumi Phos WB. siRNA transfection N27 cells were plated at 35,000 cellscm2 in antibiotic free RPMI growth medium containing 5% fetal bovine serum. SiRNA duplexes were resuspended in siRNA universal Brefeldin A protein transport buffer at 20 uM and stored in aliquots at 20 C.