CD35⁺B220⁺ cells interact with and confer CD35⁺ characteristic to stromal cells. (A) PKH26-labeled CD35⁺B220⁺ cells were cocultured with CD45⁻CD35⁻ splenic stromal cells from GFP-Tg mice for 24 hours, and the colocalization of PKH26 signals was observed and photographed with a Leica confocal microscope. Objective magnification, 63×/1.4 NA. 3-dimensional stacked images of cells with typical morphology, either round (i) or fibroblastic (ii), are shown. Cocultured cells in separate and merged color images for each staining are shown (top right, PKH26; top left, GFP; bottom left, merged image) with scale bar. (B) CD45⁻CD35⁻ splenic stromal cells cocultured with CD35⁺B220⁺ cells for 5 days were stained with anti-CD35 mAb followed by Alexa-Fluor-488-conjugated anti-mouse IgG, as described under “Materials and methods.” F-actin was visualized with Cy3-phalloidin, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were obtained with a DeltaVision RT system. Objective magnification, 60×/oil 1.4 NA. Representatives of 3 independent experiments are shown. (C) TEL-2 stromal cells were cocultured with CD35⁺B220⁺ cells from GFP-Tg mice for 24 hours within the live cell imaging module. Immunofluorescence images merged or not merged with (panel Cii) DIG images were obtained with a DeltaVision RT system. DIC-fluorescence time-lapse images from 2 to 5 hours of 24-hour incubation period (15-second gap) are shown (panel Ci, corresponding time after incubation is indicated). Objective magnification, 60×/oil 1.4 NA. Representatives of 3 independent experiments are shown. Blue bar corresponds to 30 μm, yellow bar corresponds to 20 μm, and red bar corresponds to 15 μm.