Macrophages actively secrete ferritin. (A) BMDMs were incubated with 15μM chloroquine or 80 μg/mL leupeptin for 18 hours. Ferritin was immunoprecipitated from cell lysates and medium, separated on SDS-PAGE and detected by Western blot with the anti–L-subunit antibody. For each experiment, 1 representative gel is shown of 4 similar experiments. (B) BMDM immunofluorescence of ferritin and LAMP1. Image visualization was made on a Zeiss LSM 510 META laser scanning confocal microscope with a Plan-Apochromat 63×/1.4 oil DIC lens and scan zoom 2.0, resulting in a final magnification of ×126. Alexa Fluor 488 and 568 were used as fluorochromes. Images were captured using LSM5 software program and further processed with Adobe Photoshop. (C) Electron microscopy (EM) analysis of BMDMs. L indicates lysosome; AL, autophagolysosome; M, mitochondria; ER, endoplasmic reticulum; and N, nucleus. Sections were examined with a T12 transmission electron microscope at 120keV (FEI). Image acquisition was done using a 2k CCD ultrascan CCD camera (Gatan). (D) Comparison of ratio of ferritin-Fe⁵⁵/ferritin-protein showed that the radioactive iron content of the secreted ferritin was significantly lower than that of the intracellular ferritin. BMDMs were incubated overnight with transferrin-Fe⁵⁵. Ferritin protein and Fe⁵⁵ content was determined in lysates and medium by immunoprecipitation followed either by Western blot with a purified ferritin standard curve for protein quantification or scintillation counting for Fe⁵⁵ determination.