Interaction of CD35⁺B220⁺ cells with B cells. (A) CD35⁺B220⁺ cells are not potent APCs. Colcemid-treated CD35⁺B220⁺ cells or BMDCs derived from C57BL/6J-Jcl mice were cultured with splenic CD4⁺ T cells from BALB/c mice for 4 days in quadruplicate in 96-well round-bottomed plates, and MTT reagent was added in the last 3 hours of culture. Cell proliferation was measured according to the manufacturer's instructions. Data are representative of 3 experiments yielding comparable results. The data represent the mean plus or minus SD from 3 experiments. (B) CD35⁺B220⁺ cells capture splenic B cells on their cell membrane. Splenic CD19-expressing B cells were added to CD35⁺B220⁺ cells precultured for 15 minutes on a glass-bottomed dish. DIC time-lapse images (2 minutes, 150-second gap) show dynamic changes in the shape of CD35⁺B220⁺ cells and CD19-expressing B-cell adhesion. DIC images were obtained with a DeltaVision RT system, and representatives of 3 independent experiments are shown. Photographs were taken with a 60×/oil 1.4 NA objective. (C) CD35⁺B220⁺ cells form B cell rosettes in vitro. CD35⁺B220⁺ cells and CD19-expressing B cells isolated from naive C57BL/6J-Jcl mice were cocultured for 3 days. After fixation with 4% buffered paraformaldehyde, cells were stained with anti-CD35 mAb. Merged image shows entangled CD35⁺ cell membrane of a CD35⁺B220⁺ cell with adhered B cells. Immunofluorescence images were obtained with a DeltaVision RT system, and a representative of 3 independent experiments is shown. Photographs were taken with a 60×/oil 1.4 NA objective. (D) mRNA expression of CD35⁺B220⁺ cells. Expression of different adhesion molecules was examined with RT-PCR.