Ning with anti-CD3- PE-Cy5 (eBioscience, United states), plus the samples with purity of extra than 80 have been employed for this experiment.3.3. Cell Isolation3. Components and MethodsThe fluorescent antibodies plus the corresponding isotype controls had been obtained from eBioscience (USA), and western blot antibodies have been bought from Abcam (Hong Kong). ELISA kits for IFN-, TNF-, and IL-2 was obtained from R D Co. Ltd. (USA). Ionomycin, monensin, and phorbol 12-myristate 13-acetate (PMA) have been purchased from Sigma (USA). Soluble fusion proteins CTPHBcAg18-27-Tapasin, CTP-HBcAg18-27, HBcAg18-27-Tapasin, and HBcAg18-27 were maintained in our lab (16). HLA-A2 transgenic mice (H-2Kb), six to eight weeks old, which had the murine two microglo-bulin (2m),three.1. Reagents, Mice and Fusion Proteins3.2. Mice and TreatmentsTo investigate the number of IFN- secreting cells as well as production of TNF- and IL-2 by the immunized mouse T cells, T lymphocytes (1 ?106 cells/mL) collected from immunized mice have been JAK1 Inhibitor Storage & Stability analyzed by flow cytometry. The T lymphocytes had been stimulated within the presence of ten g/mL HBcAg18-27 for six hours. Immediately after incubation for 3 hours, ionomycin (1 g/mL), monensin (1.7 g/mL), and PMA (25 g/mL) (15) were added and incubation continued for yet another 3 hours. Immediately after incubation, the wells have been washed twice with PBS; cells have been then incubated with saturating concentrations of PE conjugated anti-CD8 McAb. Right after permeabilization with Repair and Perm reagent A and B (BD Biosciences, USA), the cells was stained with FITC-labeled anti-interferon- (IFN-) McAb, APC conjugated anti-IL-2 McAb, and PE-CY7- labeled anti-TNF- for 20 minutes. AfHepat Mon. 2014;14(2):e3.4. Measurement of Function of CD8+T Cells by Intracellular Cytokine Staining (ICCS)ter two washes, the cells have been analyzed by flow cytometry (COULTER EPICS XL Flow Cytometer (Beckman)).Tang Y et al.T cells (two ?106 cells/mL) from the HLA-A2 transgenic mice harvested from immunized mice were incubated in 24-well plates at 37 C in the presence of 10 g/mL HBcAg18-27. Following 72 hours of incubation, culture supernatants were harvested and the amount of cytokines including IFN-, TNF- and IL-2 had been analyzed by ELISA kits as outlined by the manufacturer’s protocol. The concentrations of cytokines inside the samples were determined from the regular curves. Data are expressed as pg/mL. immunized mice had been cultured in Estrogen receptor Agonist Compound six-well plates at 37 as described above, except that no red blood cell lysis was performed. Just after two washes with PBS, cells have been incubated with APC-labeled anti-CD8 McAb. Annexin V ITC and Propidium Iodide (PI) staining (Invitrogen, USA) had been then performed according to the manufacturer’s instructions. The entire cell population of thrice stained positive cells among antigen-specific CD8+ T cells was analyzed by flow cytometry. T cells (2 ?106 cells/mL) from spleens harvested from immunized mice had been cultured in six-well plates at 37 C. Next, cells were collected for total RNA isolation according to the protocol for Trizol Reagent (Invitrogen, USA). cDNA was generated working with PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). Primers were created by Primer Premier five.0 based on the mRNA sequences of PI3K, Akt, and mTOR genes retrieved from GenBank, and synthesized by Sangon Biotech (Shanghai) Co., Ltd., China. The Primer sequences are shown in Table 1. Realtime PCR was performed applying SYBR remix Ex TaqTM reagents (TaKaRa, Japan) on a LightCycler (Roche Diagnostic). PCR conditions have been as follo.
