FR⁺ macrophages bind and internalize ³H-folic acid and folate conjugates. (A) Thioglycolate- (top panel) and bacteria-recruited (bottom panel) macrophages were examined for binding and internalization of ³H-folic acid under permissive (37°C) and nonpermissive (4°C) conditions for receptor-mediated endocytosis. (B,C) Confocal images show strong surface binding of folate-rhodamine after 10 minutes of incubation in thioglycolate-recruited FR⁺ macrophages. (D) Surface binding of folate-rhodamine can be blocked by coincubation with 100-fold excess of folic acid. (E,F) A fraction of bound folate-rhodamine is internalized by 2 hours at 37°C. (G) The binding and internalization of folate-rhodamine can be blocked by coincubation with 100-fold excess of folic acid. (H,I) In vivo binding and internalization of folate-rhodamine by FR⁺ macrophages. (J) In vivo binding and internalization of folate-rhodamine can be blocked by preinjection with 100-fold excess of folic acid, which indicates the uptake is FR-mediated. Scale bar represents 20 μm.