In both cases, cells were isolated and cultured as previously described. Briefly, cells were cultivated in growth medium Dulbeccos Modified Eagles Medium Hams F12 sup plemented with L glutamine gentamicin amphotericin B and 10% fetal calf serum. ACT cells were initially cul tured in autologous human serum until transplantation, that is, three to four weeks, and thereafter cryopre served. Subsequent propagation was supported by 10% FCS, whereas cells from osteoarthritic joints had growth medium supplemented with 10% FCS only. Biopsies of cartilage serving as healthy controls were taken from patients subjected to surgery due to recon struction of the anterior cruciate ligament. These patients were under the age of 35 and had no previous clinical symptoms of arthritis.
Tissue removed as part of the surgical procedure was included in the study pro vided that it had no macroscopic signs of inflammation. Reverse transcriptase polymerase chain reaction Messenger RNA from cultivated chondrocytes was extracted with Qiagen selleck chemical Direct mRNA kit. cDNA was synthesised by using SuperScript Preamplification System and treated with 0. 1 unitL E. coli RNase inhibitor at 37 C for 20 minutes. PCR was per formed in a 50 ul reaction mixture containing cDNA, 150 nM of each primer, master mix containing Taq polymerase, dNTPs, MgCl2 and buffer, and ultra pure distilled water. The PCR was performed at 94 C for 5 minutes, 94 C for 30 sec, 55 C for 30 sec and 72 C for 1 minute for a total of 30 cycles with a 10 minute final extension at 72 C. All reactions were run using a Perkin Elmer GeneAmpPCR system 2400.
The nucleotide sequences of PCR primers for human ChemR23 receptor were To selelck kinase inhibitor test the quality of mRNA, the presence of a house keeping gene transcript, adenine phosphoribosyltransfer ase, and contaminating DNA would gen erate a 800 bp fragment, whereas mRNA would gener ate a 300 bp fragment. Genomic DNA was obtained from DNA isolated from human leukocytes and was used to assess possible contamination. PCR products were analysed by the use of polyacryla mide gel, stained with SYBR safe DNA gel stain and photographed under UV light using a G BOX. The sequences of the amplicons were confirmed using BigDye Terminator v3. 1 Cycle Sequencing Kit. A total of 2 ul of each PCR product and 1 uM of each primer were processed according to the kit manual. The cycle sequencing was performed on the GeneAmp PCR Systems 9700 while the puri fication was done by capillary gel electrophoresis on the 3130XL Genetic analyzer. Immunocytochemistry To achieve the required amount of cells for in vitro experiments, cells were passaged four times.