Analysis of nitrotyrosine in thymocytes by flow cytometry.

(A) Kinetics analysis. Untreated human thymocytes cultured for several hours show a gradual increased expression of nitrotyrosine. This increase is parallel to the number of annexin V⁺ cells, except before the culture where the number of annexin V⁺cells is higher, probably due to dying cells because of the mechanical dissociation. (B) When antinitrotyrosine antibody is absorbed with free nitrotyrosine before incubation with thymocytes, the fluorescence peak is shifted to negative values (dotted line peak). The positive peak is colored in gray, and the control peak is colorless. (C) In cocultures, thymocytes incubated with TEC in presence of L-NAME (▧) show a moderate reduction of the nitrotyrosine-positive cells, but this is not observed with the MITC line used as a control thymic cell line (■).