Greetings all, We're working with culture-derived human dendritic cells and we've run into one snafu. We hope you can help. The signal with FITC-dextran is just not what we've seen in the literature. The signal is ~10x what we see with monocytes, but it does not appear as high as we expected from published reports. It does decrease slightly with maturation, as expected. The MFI of the immature population is 70, and it drops to 50 with maturation. However, the histogram of the pre-maturational population is skewed to the left. It makes me think that most cells are not taking up the dextran in the expected amounts. All of our other measures of the cells suggest the system is working well, with this one exception. We're using 40,000 MW F-Dx from Molecular Probes, P/N D-1844. Cells are incubated in culture medium with 1mg/ml F-Dx for 1 hour at 37oC, washed, and analyzed. Seems simple, but.... Are there any tricks to getting FITC-dextran to work as beautifully as in Sallusto et al. 1995 (JEM 182, 389) or Hertz et al. 2001 (JI 166, 2444)? To generate the DCs, monocytes are grown in GM-CSF plus IL-4, and pulsed every other day with the cytokines, for 6 days. They are then counted and replated with cytokines in preparation for phenotyping or functional analysis. The next day, various maturational stimuli are added. After one more day, the cells are phenotyped or T cells are added for functional analysis. In the absence of maturation the cells are CD1a+ CD83-. With 0.1ug/ml LPS >80% of the CD1a become CD83+. They increase in CD40, CD86, HLA-DR and A,B,C, etc. They have phenomenal APC function for T cells in recall responses with vaccine antigens, and mature DCs are more potent than the immature. We also see a reduction in the ability to scavenge/process antigen after maturation. Only the FITC-dextran hasn't lived up to published descriptions. Thanks in advance for any help rendered, David Duncan
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