ts of a normal diet, and the manipulation of dietary fatty acid composition may influence bone resorption, bone formation, and bone 
mass. Further elucidation of the mechanism of capric acid regulation should contribute to the discovery of novel therapeutic approaches for treating various types of inflammatory bone destruction. Materials and Methods Cell culture RAW264.7, a mouse macrophage cell line, was obtained from the ATCC. Cells were MedChemExpress 169939-93-9 cultured in 10 mm plates and maintained in high glucose Dulbecco’s Modified Eagle Medium supplemented with 2 mM glutamine, antibiotics, and 10% heat-inactivated fetal bovine serum in a 37uC humidified incubator containing 5% CO2. 5 November 2011 | Volume 6 | Issue 11 | e27739 Effect of Capric Acid on Osteoclastogenesis Reagents LPS, capric acid, L-NMMA, and a TRAP kit were purchased from Sigma-Aldrich. Antibodies specific to p-JNK, JNK, p-ERK, and ERK along with horseradish peroxidase -conjugated rabbit and mouse IgG antibodies were ” purchased from Cell Signaling Technology. Antibodies to IkB-a were purchased from Santa Cruz Biotechnology. An NO detection kit was purchased from iNtRON Biotech. An RNeasy mini kit was purchased from Qiagen Inc.. cells were incubated at 37uC for an additional 4 hr. After washing out the supernatant, the insoluble formazan product was dissolved in DMSO. Then, the optical density of the 96-well culture plates was measured using an ELISA reader at 570 nm. The optical density of formazan formed in the untreated control cells was considered 100% viability. NO analysis RAW264.7 15516710” cells were plated at a density of 56104 cells/well in 96-well plates for 12 hr and then treated with the indicated compounds for an additional 12, 18, and 24 hr. The supernatant from the cultured cells was centrifuged to remove cell debris and transferred to 96-well plates. The supernatant was then reacted using a nitric oxide detection kit. Values were calculated by measuring the absorbance at 540 nm using a plate reader. TRAP staining RAW264.7 cells were plated at a density of 56105 cells/well in 24-well plates for 12 hr and then treated with the indicated compounds for an additional 24, 48, and 72 hr. The supernatant was removed, and the cells were washed twice with PBS. Fixation solution was added to the cells and then removed with PBS. TRAP staining solution was added to the cells for 1 hr. The solution was then removed, and the cells were washed twice with PBS. Cell morphology was detected by microscopic observation. RT-PCR RAW264.7 cells were treated with each of the compounds for 24, 48, and 72 hr, followed by washing with PBS. Total RNA was isolated with an RNeasy Mini kit, and the total RNA concentration was detected using a spectrophotometer. Total RNA was converted to cDNA with cDNA Synthesis Master Mix. PCR was performed using Maxim PCR Premix. The PCR primers were as follows: TRAP- F; 59-TCC CCT GGT ATG TGC TGG-39 and R; 59GCA TTT TGG GCT GCT GA-39, iNOS- F; 59-GCA GAA TGT GAC CAT CAT GG-39 and R; 59-ACA ACC TTG GTG TTG AAG GC-39, MCP-1- F; 59-GAA GGA ATG GGT CCA GAC AT-39 and R; 59-ACG GGT CAA CTT CAC ATT CA-39, 6 November 2011 | Volume 6 | Issue 11 | e27739 3–2,5-diphenyltetrazolium bromide assay Cell viability was determined using a colorimetric 3–2,5-diphenyltetrazolium bromide assay. RAW264.7 cells were cultured in 96-well plates for 24, 48, and 72 hr after LPS treatment with or without capric acid. MTT solution was added, and the Effect of Capric Acid on Osteoclastogenesis COX-2- F;
