Figure 3.

The dynamics of CD4⁺ T memory (Tm) cell proliferation in co-culture and transwell assay. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled CD4⁺ Tm cells were cultured in the presence of interleukin (IL)-7/IL-15 and in contact with monocytes or myeloid dendritic cells (MDCs) (co-culture assay) or separated from monocytes or MDCs (transwell assay) for 7 days. Cultured cells were labelled with anti-CD3-phycoerythrin (PE), CD11c-allophycocyanin and CD45-peridinin-chorophyll-protein (PerCp) monoclonal antibodies (mAbs) and analysed by flow cytometry. Regions R1, R2 and R3 were created to define live CD45⁺ cells, CD3⁺ T cells or TruCOUNT^TM beads, respectively (see Fig. 1). (a) Histograms show the fluorescence profile of CFSE-labelled CD3⁺ T cells (corresponding to CD4⁺ Tm cells). The numbers (0–3) above the histograms denote division number (see Fig. 2). (b) The absolute number of CD4⁺ Tm cells recovered in co-culture and transwell assay were analysed by TruCOUNT^TM assay (see Fig. 1). Data are displayed as the total number of CD3⁺ T cells/well [corresponding to CD4⁺ Tm cells/well; mean ± standard error of the mean (SEM) for triplicate measurements] of one representative experiment of four. APCs, antigen-presenting cells; Mo, monocytes.