1. 0 ug of total RNA was sub jected to oligo dT primed RT with ReverTra Ace Kit. Semi quantitative PCR was carried out with DNA polymerase by using specific primers which amplify inhibitor Idelalisib 349 bp product for b actin. The amplified PCR products were resolved by 2% agarose gel electrophoresis. Real time PCR was performed for a quantitative analy sis of iNOS, IL 1b and TNF a mRNA expression using SYBR Green real time PCR Master Mix on an MX3000P real time PCR system. The fol lowing primers were used, 101 bp product for b actin. Relative gene expression was calculated by the 2 CT method. Cell viability assay Cell viability was measured by quantitative colorimetric assay with MTT, showing the mito chondrial activity of living cells.
BV 2 cells in 96 well plates were pretreated with various concentrations of ATL for 30 min and incubated with or without LPS for 24 h in the continued presence of ATL. Upon termina tion of the experiments, the culture media were aspi rated and MTT was added to cells Inhibitors,Modulators,Libraries and then incubated at 37 C for 4 h. The supernatant was aspi rated and dimethyl sulfoxide was added to the wells. Insoluble crystals were dissolved by mixing and the plates were read on an automated Tecan Sun rise absorbance reader, using a test wavelength Inhibitors,Modulators,Libraries of 570 nm and a reference wavelength of 630 nm. Nitrite measurements Production of NO was determined by measuring the level of accumulated nitrite, a metabolite of NO in the culture supernatant using Griess reagent. After 24 h of treatment with LPS with or with out ATL, the culture supernatants were collected and mixed with an equal volume of Griess reagent in 96 well culture plates and incubated at room temperature for 10 min.
The absorbance was measured at 540 nm and nitrite concentrations were calculated by reference to a standard curve generated by known concentrations of sodium nitrite. ELISA for IL 1b and Inhibitors,Modulators,Libraries TNF a BV 2 cells in 24 well plates were stimulated for 24 h, and then culture supernatants were harvested. Levels of IL 1b and TNF a in 100 ul medium were measured by commercial ELISA kits according to Inhibitors,Modulators,Libraries the manufacturers instructions. Immunofluorescence confocal microscopy For the detection of intracellular location of NF B p65, BV 2 cells were cultured on sterile glass cover slips in 24 well plates and treated with ATL and LPS as described above. At various times after the LPS treat ment, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0. 1% Triton X 100 in Inhibitors,Modulators,Libraries PBS. After rinsing, cells were blocked with selleck 3% BSA in PBS for 1 h and incubated with rabbit anti NF B p65 antibodies overnight at 4 C. After washing, cells were incubated with FITC conjugated goat anti rabbit IgG for 1 h and counterstained with 4, 6 diamidino 2 phenylindole for the identification of nuclei.