ROS production correlates with expression of FR on macrophages. (A) FR⁺ macrophages actively produce ROS. Macrophages isolated from bacteria-treated mice were simultaneously incubated with (1) carboxy-H₂DCFDA to image ROS (green), (2) folate-rhodamine to image FR (red), and (3) anti-F4/80 to stain macrophages (blue) and then examined by confocal microscopy. The overlay of all the 3 colors within the same microscope field is also shown. (B) Cells collected from the peritoneal cavities of mice injected 3 days earlier with different doses of bacteria were analyzed by flow cytometry for the binding of folate-FITC and the production of ROS. The fraction of FR⁺ and ROS⁺ cells is indicated. (C) The percentage of ROS⁺ cells correlate linearly with the percentage of FR⁺ macrophages. Scale bar represents 20 μm.