PU.1-induced DCs are capable of maturation in response to LPS. E26ts21-PUER clones were cultured for 72 hours in the absence (A,D,G) or presence (B,E,H) of 1 μM βE, or treated for the last 48 hours with 10 ng/mL LPS in addition (C,F,I). (A-C) FACS analysis of MHCII expression on the cell surface. (D-F) Immunofluorescence detection of MHCII by confocal microscopy. Note weak surface expression and round morphology of myeloblasts (D), lysosomal MHCII vesicles in veiled immature DCs (E), and strong surface expression on prolonged membrane protrusion (×40) typical of mature DCs (F). Images in panels D-F were obtained using a Zeiss Axiovert-200 confocal microscope equipped with a 63×/1.4 objective lens and an integrated camera (Zeiss, Jena, Germany). LSM 510 version 3.2 software was used for image acquisition. Contrast and exposure were enhanced through Adobe Photoshop 5.0 using equal treatment on all panels. (G-H) Phase-contrast images of the cells in culture were obtained with a confocal Zeiss Axiovert 200 microscope equipped with a motorized 63×/1.4 objective lens. Images were acquired with LSM10 software version 3.2 (Zeiss, Jena, Germany). Note the formation of clusters typical of DC maturation after LPS stimulation (I). (J) Mixed lymphocyte reaction with PUER (▪) or MafB clones (▦) induced (ind.) to differentiate by βE addition or temperature shift, respectively, and in the absence or presence of LPS stimulation as indicated, at a stimulating cell–splenocyte ratio of 0.3 and expressed as proliferation index. Data are representative of 3 independent experiments.