Identification of CD19⁻CD11c⁻CD35⁺B220⁺ cells in mouse spleen. (A) Flow cytometric analysis of spleen cells from C57BL/6J-Jcl mice and TNF-KO mice. Whole spleen cells stained with indicated mAbs were analyzed. Gating of CD19⁻ and CD11c⁻ cells enabled the identification of CD35⁺ cells in both strains. Frequencies of CD35⁺B220⁺ cells within CD19⁻ and CD11c⁻ populations of splenocytes are shown at the corner of respective quadrants. (B) Isolated CD35⁺B220⁺ cells show the MHC class II⁺ phenotype. The phenotype of the isolated cell population, termed CD35⁺B220⁺ cells, was characterized by double color staining for MHC class II and CD35. Data are representative of 3 independent experiments. (C) Morphology of CD35⁺B220⁺ cells. Isolated CD35⁺B220⁺ cells from GFP-Tg mice were added dropwise onto a glass slide, air-dried, and fixed with 4% buffered paraformaldehyde. GFP-mediated signals were observed and photographed with a Leica confocal microscope. Photographs were taken with a 63×/1.4 NA objective.