Figure 8

Ex vivo detection of Myhc-α-reactive cells by IA^k/Myhc-α 334-352 tetramers and dextramers. (A) LNC. CD4 cells were isolated from LNC harvested from immunized A/J mice and, after treatment with NASE, cells were stained with Myhc-α 334-352 or control tetramers and dextramers, followed by anti-CD4 and 7-AAD and anti-CD25 or anti-CD44. After acquiring the cells by FC, percentages of tet⁺ and dext⁺ live (7-AAD^-) cells were determined in CD4, CD25 and CD44 subsets. Flow cytometric plots and mean ± SEM values obtained from four individual experiments, each involving 3 to 5 mice, are depicted in top and bottom left panels, respectively. (B) MNC from hearts. Groups of A/J mice were immunized with Myhc-α 334-352 and 21 days later, mice were sacrificed and hearts were collected to harvest MNC by percoll density gradient centrifugation. After staining the cells with Myhc-α 334-352 or control tetramers or dextramers and anti-CD4 and 7-AAD, cells were acquired by FC, and the percentages of tet⁺ and dext⁺ cells were enumerated in the live CD4 subset. Representative data from two individual experiments each involving 7 mice, are shown. Control, RNase 43-56.