Figure 1.
Figure 1. Generation of rat bone marrow-derived immature myeloid DCs. Rat bone marrow cultures were sequentially treated with Flt3-ligand + GM-CSF for 7 days and then GM-CSF + IL-4 for 3 days. Nonadherent cells were then harvested and characterized by using hematoxylin-eosin staining (A,E), immunocytofluorescence (B,F,D,H), electron microscopy (C,G), or FACS analysis (I). (A,D) Cells stained with hematoxylin-eosin show a round irregular morphology (A) or bear multiple dendrites (E). (B,F) Round irregular cells (B) as well as process-bearing cells (F) display strong immunostaining against MHC class II molecules. (C,G) Electron microscopy allows round irregular cells with numerous phagosomes and phagolysosomes (C) to be distinguished from process-bearing cells showing a less developed endosomal compartment (G). (D,H) The phagocytic activity of bone marrow-derived DCs was evaluated by adding, in the culture medium, fluorescent latex microspheres of 1-μm diameter for 24 hours. Using confocal microscopy, fluorescent microspheres (red) are observed in the cytoplasm of MHC class II+ round irregular DCs (D) or MHC class II+ (green) process-bearing DCs (H). The inset in panel D shows a high magnification view of MHC class II+ endocytic vesicles (green) having internalized fluorescent microspheres (red). (I) FACS analysis shows that cells uniformly express OX62, CD11c, and OX42, indicating they are myeloid dendritic cells. They also show low-to-intermediate levels of MHC class II molecules, CD80, and CD86, indicating they are immature DCs. (J) When stimulated with LPS, DCs acquire phenotypic features of mature DCs because they express high membranous levels of CD11c, MHC class II, and CD86 molecules as compared with control staining (gray curve). For results of FACS analysis, in each quadrant the percentage of cells is shown displaying fluorescence intensity above the background level obtained with a control antibody (gray curve), the mean fluorescence intensity (MFI), and the factor of MFI increase as compared with control MFI. Scale bars: 8 μm (A,E), 4 μm (B,F), 2 μm (C,G), 2 μm (D), 1.5 μm (inset in D), and 2 μm (H). Data shown are representative of at least 3 experiments.

Generation of rat bone marrow-derived immature myeloid DCs. Rat bone marrow cultures were sequentially treated with Flt3-ligand + GM-CSF for 7 days and then GM-CSF + IL-4 for 3 days. Nonadherent cells were then harvested and characterized by using hematoxylin-eosin staining (A,E), immunocytofluorescence (B,F,D,H), electron microscopy (C,G), or FACS analysis (I). (A,D) Cells stained with hematoxylin-eosin show a round irregular morphology (A) or bear multiple dendrites (E). (B,F) Round irregular cells (B) as well as process-bearing cells (F) display strong immunostaining against MHC class II molecules. (C,G) Electron microscopy allows round irregular cells with numerous phagosomes and phagolysosomes (C) to be distinguished from process-bearing cells showing a less developed endosomal compartment (G). (D,H) The phagocytic activity of bone marrow-derived DCs was evaluated by adding, in the culture medium, fluorescent latex microspheres of 1-μm diameter for 24 hours. Using confocal microscopy, fluorescent microspheres (red) are observed in the cytoplasm of MHC class II+ round irregular DCs (D) or MHC class II+ (green) process-bearing DCs (H). The inset in panel D shows a high magnification view of MHC class II+ endocytic vesicles (green) having internalized fluorescent microspheres (red). (I) FACS analysis shows that cells uniformly express OX62, CD11c, and OX42, indicating they are myeloid dendritic cells. They also show low-to-intermediate levels of MHC class II molecules, CD80, and CD86, indicating they are immature DCs. (J) When stimulated with LPS, DCs acquire phenotypic features of mature DCs because they express high membranous levels of CD11c, MHC class II, and CD86 molecules as compared with control staining (gray curve). For results of FACS analysis, in each quadrant the percentage of cells is shown displaying fluorescence intensity above the background level obtained with a control antibody (gray curve), the mean fluorescence intensity (MFI), and the factor of MFI increase as compared with control MFI. Scale bars: 8 μm (A,E), 4 μm (B,F), 2 μm (C,G), 2 μm (D), 1.5 μm (inset in D), and 2 μm (H). Data shown are representative of at least 3 experiments.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal