Ondrocytes Chondrochondrocytes. cytes were obtained from the femoral condyle and theOndrocytes Chondrochondrocytes. cytes were obtained

Ondrocytes Chondrochondrocytes. cytes were obtained from the femoral condyle and the
Ondrocytes Chondrochondrocytes. cytes were obtained from the femoral condyle and the tibial plateau from osteoarthritis (OA) patients and cultured in monolayers. First passage chondrocytes were used in subsequent experiments. (a) Dose-dependent and (b) time-dependent transduction of Tat-SOD into chondrocytes. Transduction of Tat-SOD into the cells was analyzed by western blotting with a rabbit anti-polyhistidine IgG. Tat-SOD (1 to 7 ) and control SOD were added to the culture medium for 1 hour (a), or 3 of Tat-SOD and control SOD were added to the culture medium for 1 to 9 hours (b). (c) Localization of transduced Tat-SOD protein. After FITC-labeled Tat-SOD (3 ) was transduced into chondrocytes, the cells were washed with PBS and immediately observed by fluorescence microscopy (?00 original magnification; inset, ?00 original magnification). Data are representative of four samples from different donors. (d) The specific activities of SOD in cultured chondrocytes treated with Tat-SOD; 1 to 7 of Tat-SOD and control SOD were added to the culture medium for 1 hours. Bars represent the mean ?standard error of the mean obtained from duplicate experiments from three donors. Asterisks denote p < 0.05 compared to control.Chondrocyte culture Cartilage samples were obtained from the femoral condyle and tibial plateau of OA patients at the time of joint replacement surgery. All cartilage samples were procured after obtaining oral informed consent from the patients and institutional approval. Chondrocytes from articular cartilage were cultured in monolayer as described previously [18]. After about seven days, confluent chondrocytes were split once, plated and these first passage adherent chondrocytes were used in subsequent experiments. For cartilage explant culture, full-thickness cartilage slices were obtained above the subchondral bone from a relatively lesion-free area of the femoral condyle of OA patients. Each slice was cut further and a piece of approximately 2 mm width by 5 mm length by full thickness was cultured in a 48-well culture plate in 200 per well of the same medium in which monolayer chondrocytes were cultured. Explants were incubated in the medium forthree days before protein transduction to allow them to stabilize in in vitro conditions.Transduction of Tat-SOD into chondrocytes For the transduction of Tat-SOD protein into the monolayer cultured chondrocytes, cells were seeded at 5 ?105/well in six well plates. Then, the culture medium was replaced with 1 ml of fresh serum free DMEM containing various concentrations of ICG-001 web fusion protein. For the transduction of Tat-SOD into the cartilage explant culture, the culture medium was replaced with 200 of fresh serum free DMEM containing various concentrations of fusion proteins. The transduction procedures were the same for control SOD and Tat-GFP proteins. Western blot analysis Monolayer cultured chondrocytes were extensively washed after protein transduction, and trypsinized. Cellular proteins were extracted in lysis buffer containing 50 mM sodium ace-Page 3 of(page number not for citation purposes)Arthritis Research TherapyVol 8 NoKim et al.FigureTransduction of transactivator of transcription (Tat)-superoxide PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25962748 dismutase (SOD) fusion protein into explant cultured chondrocytes Chondrocytes chondrocytes. were obtained from a relatively lesion-free area of femoral condyles in OA patients and cultured in explants. (a) Transduction of Tat-SOD into the cells was analyzed by western b.