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Fig. 1. Endogenous GM-CSF is undetectable in IL-4-DC. (A) Detection of intracellular GM-CSF in monocytes (freshly isolated or cultured for 1 day and 7 days in the presence of IL-4) before and after stimulation with LPS. Relevant cells were surface stained for the expression of CD11c (FL2), permeabilized, stained for intracellular GM-CSF (FL1). Quadrants were set after staining with isotype-matched control mAbs. The proportion of positive cells are given in each quadrant. One representative experiment of three is shown. (B) Specificity and sensitivity of intracellular GM-CSF staining was demonstrated by surface staining fresh monocytes (stimulated with medium or LPS) with CD11c followed by staining for GM-CSF without permeabilization. (C) Detection of GM-CSF transcripts by RT-PCR. Total RNA was extracted from relevant cells, 500 ng (i) and 2000 ng (ii) RNA were used to analyse the expression of mRNA for GM-CSF and ß-actin. Data are representative of three similar experiments. (D) Yields of immature and mature cells. The numbers of monocytes plated on day 0 was set equal to 100%. Percentages indicate the numbers of recovered cells on day 7 for immature and on day 9 of culture for mature cells. Data shown are from three separate experiments; error bars indicate s.d. (E) Phase contrast microscopy of TNF--stimulated (48 hours) mature cells. Note the numerous veiled processes in IL-4-DC and GM-IL-4-DC. Magnification x300.

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