Fig 1.

Visualizing K^d-specific B cells in naïve C57BL/6 mice with fluorescent K^d-tetramers. (a) The percentage of B220⁺ B cells binding to Streptavidin-phycoerythrin (SAV-PE), Streptavidin-allophycocyanin (SAV-AP), or both (Left), compared to binding of K^d tetramer-PE or K^d tetramer-APC or both (Right). (b) To enhance detection of alloreactive B cells, single-cell suspensions of spleen and lymph node cells of naïve C57BL/6 mice were stained with either the combination of K^d-PE and K^d-APC or SAV-PE and SAV-APC, and then enriched by anti-PE and anti-APC microbead positive selection. The percentage (following enrichment) and total numbers of B220+ B cells in a naïve C57BL/6 mouse binding only to K^d tetramer-PE or K^d tetramer-APC or to both (K^d Tet-DP) were determined. (N=4). (c) K^d Tet-DP (K^d-PE and K^d-FITC tetramers) B cells display minimal binding to irrelevant molecules, K^b Tet⁺ APC or SAV-APC. Data are the mean and SEM of 4 different individuals. (d) K^d Tet DP B cells are reduced in MD4 HEL BCR transgenic mice. Inguinal LNs from MD4 BCR transgenic mice or transgene negative littermates were collected and stained for K^d Tet DP B cells.