Spleens were harvested under sterile conditions. Single-cell suspensions were prepared, and red cells were removed using ACK lysing buffer. Two million splenocytes were incubated for 30 min on ice in staining media (1% FBS in PBS) with the relevant Abs and then washed with PBS. For intracellular staining of nitrotyrosine, cells were labeled with anti-CD11b-APC, anti-Ly6C-FITC, and anti-Ly6G-PE, fixed and permeabilized with Cytofix/Cytoperm Buffer (BD Biosciences) and washed with a 1× PermWash solution (BD Biosciences). The cells were incubated with rabbit polyclonal anti-nitrotyrosine antibody for 1 hr on ice. After washing, the cells were incubated with the secondary detection reagents, goat anti-rabbit IgG (H+L)-Alexa Fluor 647 for 45 min on ice. After washing, the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and were analyzed using FlowJo software (Tree Star, Ashland, OR).