FIG. 7.

Ferryl hemoglobin is ingested by macrophages. (A–D) Macrophages grown on coverslips were exposed to oxyHb (10 μM), ferrylHb (10 μM), or growth medium for 12 h. Cells were stained with Hoechst 33258 for DNA (blue), an anti-LAMP-1 antibody with Alexa Fluor 488 secondary antibody for lysosomes (green), an anti-ferrylHb antibody with Alexa Flour 568 secondary antibody for ferrylHb (yellow), and iFluor 647 phalloidin for cytoskeleton (red). (B) Fluorescence intensity for ferrylHb staining was calculated using ImageJ software (n = 5). (C) Actin-lined phagocytosis of the ferrylHb was shown. (A, B) Multicolor confocal imaging was acquired with a Leica TCS SP8 microscope. (D) Localization of the ferrylHb (for better contrast the color of ferrylHb was changed from yellow to red) inside the lysosomes (green) was demonstrated using Leica TCS SP8 gated STED-CW nanoscopy. Images were deconvolved using Huygens Professional software. Representative images are shown (n = 5). Scale bars shown in the images represent 0.5, 0.75, and 10 μm. (E) Macrophages were exposed to ferrylHb (10 μM) or growth medium for 24 h. Six micrograms of protein of isolated lysosomes was analyzed by Western blot. Expressions of LAMP-1, Hb, and GAPDH were assessed (n = 3). ***p < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMP-1, lysosomal-associated membrane protein 1. Color images are available online.