Dynasore

Monocytes were obtained by gradient centrifugation of peripheral blood mononuclear cells (PBMCs) and cultured as described.33 At day 3, 25 ng/mL of granulocyte colony‐stimulating factor (Invitrogen Corporation, Carlsbad, CA, USA) were added, and macrophages were selected by adhesion. Medium and all reagents were endotoxin free.
Macrophage lineage characterization was based on morphological study and expression of the monocytes‐macrophages lineage marker, CD14 (see below).
To block dynamin‐dependent endocytosis,34in vitro differentiated macrophages were treated with 40 and 400 μM of dynasore (Enzo Life Sciences, Lausen, Switzerland) for 30 minutes and processed for immunoflurescence.
Cells were also treated for 1‐24 hours with 700 nM of Hepc (Peptide Institute, Minoh‐shi Osaka, Japan) and subjected to immunofluorescence.
In specific experiments, cells were preincubated for 18 hours with 0.25 µM of dexamethasone phosphate (Dx; Sigma‐Aldrich, St. Louis, MO, USA), a known stimulator of CD163 expression and hemoglobin (Hb) uptake by macrophages,35 and then incubated for 6 hours with 20 μg/mL of bovine Hb (Sigma‐Aldrich). Donor macrophages were also treated with aged red blood cells as reported.36 Cells were harvested and processed for immunofluorescence.
Cellular ferritin content and iron in the medium were assayed after overnight incubation as specified below.

J774 cells stably expressing Myc-tagged proteins were washed in ice-cold HBSS and incubated for 30 min on ice in the presence of an ∼20-fold excess amount of IgG-opsonized latex beads incubated for 5 min at 30°C to initiate phagocytosis. The cells were washed three times in ice-cold HBSS and incubated for 20 min on ice in the presence of Alexa Fluor 647 to stain the beads outside the cells to distinguish them from engulfed beads. The cells were then washed in ice-cold HBSS and incubated in HBSS containing DMSO, dynasore (final 50 µM, Enzo Life Sciences, Farmingdale, NY), MiTMAB (final 20 µM, Sigma-Aldrich), or Pitstop 2 for the indicated times at 30°C in the presence of cytochalasin B to inhibit the formation of new phagosomes. Following incubation, the cells were washed in PBS and fixed with 100% methanol for 7 min at −20°C. Subsequently, the cells were incubated with 2% BSA/PBS for 30 min at 25°C, treated with anti-Myc and anti-LAMP1 antibodies, and stained with Alexa Fluor 488-conjugated goat antimouse and Alexa Fluor 594-conjugated goat antirat secondary antibodies (Invitrogen), respectively. Images were captured on an LSM780 microscope. More than 30 phagosomes were counted for each experiment and categorized into two types (Myc- or LAMP1-positive phagosome or unlabeled phagosome) based on the presence or absence of a detectable Alexa Fluor fluorescence signal.