Short-term development of CMRF-44⁺⁺ DC in culture is augmented by additional signals. Freshly isolated PB lineage marker negative cells were prepared (as described in Materials and Methods) and were cultured for 40 hours in media alone or with additional cytokines as shown. The plots are gated on live cells (as assessed by forward and 90° light scatter characteristics) which expressed HLA-DR. A total of 10,000 events were collected in each case using identical gating criteria. The relative mean fluorescence intensity (MFI) of CMRF-44 labeling in each case was calculated using the formula: MFI CMRF-44 (media + cytokine) - MFI (control Ab)/MFI CMRF-44 (media alone) - MFI (control Ab) and expressed as a percentage. Additional GM-CSF or TNFα both increased the number of viable cells by up to 20% compared with media alone. Quadrants are placed as dictated by isotype matched control antibody (x axis) and at the level of class II expressed on these cells before culture (y axis). Percentages of cells falling into each quadrant are shown. The three-dimensional plots depict the same data and allows a comparison of the cytokine effect at each stage of DC development.