Daughter cells contain half the fluorescent intensity of the parent cell. Figure 3 CD8 + T cells cytolytic activity in the immunized mice as demonstrated by IFN-γ intracellular staining. Two weeks after the last HCV vaccine immunization,
cultured splenocytes were unstimulated (A), stimulated with CE1E2 protein (B), core peptide (C), or vaccinia HCV poly (D). Cells were cultured for 18 hrs in the presence of brefeldin A then stained intracellularly with anti-IFN-γ antibody and surface stained with anti-CD3+ and anti-CD8+ antibodies to be analyzed by flow cytometry. Percentages in the upper right quadrant represent the frequency of CD3+8+ T lymphocytes expressing IFN-γ. The P value for significant differences was < 0.05. Figure 4 Detection of CD4 + and CD8 + T lymphocyte responses to HCV vaccine in immunized mice using IFN-γ ELISPOT assay. ELISPOT counts (spot-forming units [SFUs]/1 × https://www.selleckchem.com/products/pifithrin-alpha.html 106) in response to core, E1 and E2 protein, Core peptides, or vaccinia HCV poly. Spot forming cell
(SFC) frequencies are shown after subtraction of background with unstimulated cells or empty vaccinia stimulated cells. Cells were incubated with core, E1 and E2 protein, Core peptides, or vaccinia HCV poly for 48 hrs before measuring IFN-γ ELISPOT responses. Spot forming cell (SFC) frequency Selleckchem Blasticidin S per million cells is indicated for each immunized and non-immunized donor mice. The P value was < 0.05. Flow cytometric analysis of recipient mouse tissues To study the splenocyte kinetics Methocarbamol in the HCV transgenic mice and to indirectly evaluate the immune response
generated after HCV vaccination, splenocytes from the immunized and control mice were collected and labeled with CFSE before performing the adoptive transfer. CFSE labeled splenocytes were then confirmed by immunofluorescent microscopy (Figure 5). These cells were injected intravenously in transgenic and control mice and tracked down in the blood in vivo after 24 hrs. Seven days after the adoptive transfer, recipient mice were euthanized. The location and number of transferred cells were detected by flow cytometry in blood, lymph nodes, spleens and livers of recipient mice. Figure 5 Immunofluoresent analysis of CFSE labeled splenocytes before injection. A) CFSE unlabeled splenocytes showing no CFSE staining. B) CFSE labeled splenocytes showing green fluorescent cells. Scale bar = 50 μm. All groups of recipient mice had similar percentages of donor CD4+ and CD8+ T cells at 24 hrs post-adoptive transfer, indicating that all groups received similar amounts of donor splenocytes (Figure 6a). Seven days after the adoptive transfer, the percentage of the donor CD4+ and CD8+ T cells in the blood differed between the recipient mice receiving immunized and non-immunized donor cells (Figure 6b).