Journal: Journal of Virology
Article Title: Ex Vivo Phenotype and Frequency of Influenza Virus-Specific CD4 Memory T Cells
Figure Lengend Snippet: Frequencies of circulating DR1-HA tetramer-positive cells ex vivo. (A) Example of ex vivo DR1-HA tetramer staining of CD4 T cells before and after magnetic bead enrichment for PE-positive cells. PBMCs were stained with PE-conjugated DR1-HA tetramer, APC-conjugated anti-CD4 antibody, PerCP-conjugated anti-CD14 and -CD19 antibodies to exclude monocytes and B cells, respectively, and Via-Probe to exclude dead cells. Cells were incubated with anti-PE microbeads, and PE-positive cells were selected (enriched) on a magnetic column. Plots are gated on CD4 + CD14 − CD19 − Via-Probe − cells. The plot on the left shows DR1-HA tetramer staining of CD4 + cells before enrichment, and the plot on the right shows the same cell population after enrichment. The frequency of DR1-HA tetramer-positive cells after enrichment was calculated by dividing the number of CD4 + tetramer + cells after enrichment by the input number of CD4 cells. In this example, the input number of CD4 cells was 759,340 and the output number of CD4 + DR1-HA + cells was 46. (B) Reproducibility of the frequency estimations of DR1-HA tetramer-positive cells in six healthy DR1-positive individuals from a single time point. Between two and eight replicate tetramer staining assays were performed on a single PBMC collection from each subject. The frequency of tetramer-positive cells was analyzed as described above. The horizontal bar represents the mean tetramer-positive frequency for each individual. (C) Longitudinal analysis of DR1-HA tetramer-positive cells in two DR1-positive individuals. Four PBMC samples were obtained over a 12-month period and analyzed as described above. The mean frequency of DR1-HA tetramer-positive cells at each time point is plotted for subjects 1 and 4.
Article Snippet: All results shown were obtained using the DR1-HA tetramer purchased from Beckman Coulter, with the exception of the results for the first three time points for subjects 1 and 4 shown in Fig. , which were obtained using the DR1-HA tetramer constructed as previously described ( ).
Techniques: Ex Vivo, Staining, Incubation