Transfection was performed by 2 electroporation shocks at 1.4-1.6 KV using an electroporation apparatus (BTX Inc., San Diego, CA). The transfected cells were incubated in IMDM (Sigma-Aldrich, St. Louis, MO) P5091 mw containing 10% FCS (Life Technologies Laboratories, Grand Island, NY) and 50 μ g/mL penicillin-gentamicin. At 65 hrs after transfection the cells were harvested, lysed in lysis buffer (25 mmol/L Tris base, 2.5 mmol/L mercaptoethanol, and 1% Triton-X100),

sonicated, and subjected to protein purification using the Talon affinity resin kit as described before. The purity of the protein was verified by mass spectrometry, and protein with ~85% purity SCH727965 research buy was used for immunization. Immunization strategy of donor mice Eight donor mice were immunized with a HCV vaccine containing pVAX-HCV Core, E1 and E2 DNA (100 μg); Core, E1 and E2 protein (25 μg) in PBS solution and montanide (50 μl) ISA-51 (Seppic Inc., Fairfield, NJ) was used as adjuvant. Mice were immunized three times with 100 μl of the vaccine and boosted twice intramuscularly in the quadriceps major with two weeks intervals between each boost. Eight wild-type non-immunized mice were injected with PBS solution and montanide ISA-51 alone and used as a negative control. After each immunization, the humoral immune response was assessed

by Pictilisib an IgG ELISA using mouse sera. The cellular immune response was assessed using PBMCs isolated from the whole blood after the first immunizations and using PBMCs isolated from splenocytes after the last immunization. The mice were anesthetized with 50 Somnotal (MTC Pharmaceuticals, Cambridge, ON, Canada), sacrificed, and blood and spleens were Selleck Hydroxychloroquine collected. Preparation of lymphocytes

from donor mouse spleens Donor mice were sacrificed using anesthetic, and spleens were removed and placed in tubes containing sterile PBS. Lymphocytes were prepared as a cell suspension by gently pressing organ segments through a fine plastic cell strainer using a plastic pipette; then, 10 ml of PBS was added to pass cells through the mesh. The spleen cell suspensions were depleted of red blood cells (RBC) using RBCs lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA). The cellular suspension was washed three times by adding 0.1% BSA in PBS and centrifuged at 1600 rpm at 4°C for 5 min. The cells were counted and divided into 2 parts: cells for CFSE labeling, which were used for injection and CFSE proliferation assay, and cells for CTL and ELISPOT assays used to assess the immune response. ELISA To assess the antibody titer against the HCV vaccine, mice were bled at different points after the immunizations and the serum was collected. Serum levels of hepatitis C-specific antibodies were measured using the HCV recombinant core/E1/E2 polyprotein as a capture molecule and a mouse-specific monoclonal antibody-horseradish peroxidase (HRP) conjugate detection system. EIA/RIA Stripwell™ plates (Corning CoStar Inc.