Tiam Mana Saffari

112 CHAPTER 6 Blood collection At one and two weeks postoperatively, rats were anesthetized using isof lurane and blood was drawn from the tail vein to measure CD4 and CD8 levels. Rats were positioned on a heat pad to dilate the tail vein during this experiment. Flowcytometry Per sample, 2 ml of blood was collected in heparin tubes. Within an hour, blood was transferred to vials containing 1 ml of PBS. After decantation of Ficoll-Paque, vials were centrifuged at room temperature at 2000 RPM for 30 minutes to separate the buffy coat. The buffy coat of peripheral blood lymphocytes (PBL) was transferred to a vial containing 1 ml of FACS buffer. This suspension was centrifuged at 4°C at 1500 RPM for 5 minutes, the supernatant was discarded and the pellet was used for the next steps. For detection of CD4 and CD8 cells, PE-anti-CD4 monoclonal antibody (clone W3/25, 1:100, ThermoFisher, IL, USA) and FITC anti-CD8 alpha monoclonal antibody (clone OX-8, 1:100, ThermoFisher, IL, USA) was used, respectively. Samples were incubated on ice for 30 minutes, washed with FACS buffer and centrifuged at 4°C at 1500 RPM for 5 minutes. The supernatant was discarded and washing was performed three times. Cells were fixated with 500 ul of 1% PFA and analyzed using flow cytometry (FACScan, LSR II, BD™ ). Messenger ribonucleic acid (mRNA) quantitative real time PCR (RT-qPCR) All chemicals and primers for RNA extraction and analysis were purchased from Millipore Sigma (St. Louis, MO) unless stated otherwise. After sacrifice, nerve grafts within the sutures were collected in 1 mL Trizol (TRI Reagent, Sigma, USA), placed on ice immediately and stored at -80°C. Samples were mechanically homogenized (IKA Ultra Turrax T8 Homogenizer, NC, USA) and stored on ice for 15 minutes before manual extraction of RNA. To 1 mL of Trizol suspension, 200 µl of chloroform was added. Samples were vortexed, spun down at 4°C for 15 minutes at 12,000g and the aqueous phase was carefully transferred to a new tube. After adding 500 µl of isopropyl alcohol (2-propanol), samples were precipitated at -20°C for one hour and spun down at 4°C for 20 minutes at 12,000g. The supernatant was removed and the RNA pellet was washed with 75% Ethanol (EtOH). Samples were spun down for 20