Figure 2

Optimization of reaction conditions for IA^s/PLP 139-151 dextramer staining. (A) Duration of staining. PLP139-151-reactive cells generated from immunized mice were stained with PE-labeled tetramers or dextramers for PLP 139-151 and their controls. After incubating for indicated time-points, cells were stained with anti-CD4 and 7-AAD and acquired by FC; percentages of dext⁺ or tet⁺ cells were determined in the live (7-AAD^-) CD4 subset. (B) Amount of dextramers. PLP 139-151-reactive cells were stained with the indicated concentrations of PLP 139-151 or control dextramers, and percentages of live dext⁺ cells were determined in CD4 subset as above. The amount of biotinylated IA^s protein present in the 4 x-concentration was 1.72 μg and the protein concentration was halved serially in the remaining dilutions as shown. (C) Freeze-thaw cycling. PLP 139-151 or control tetramers and dextramers were thawed upto six times after freezing at -80°C for a minimum of half an hour between cycles and the reagents were used to stain PLP-reactive cells as above. Percentages of tet⁺ or dext⁺ cells were evaluated in the live CD4 subset. Control, TMEV 70-86. (D) Cell density. PLP 139-151 or control dextramers were used to stain PLP-reactive cells at the indicated cell numbers as above, and the percentages of dext⁺ cells were evaluated in the live CD4 subset. Mean ± SEM values obtained from three individual experiments each involving two mice are shown.