Figure 2.

Endocytic properties of wild-type (WT) and iron-sensitive (IS) lines of RAW 264.7 macrophages. WT and IS cells were incubated in culture conditions with growth medium supplemented with 50 µM of calcein green (CALG) given as either CALG-Fe (1:1 complexes) for 30 min or CALG-V (1:2 complexes) for 4 h or with opsonized erythrocytes for 1 h, washed and analysed by phase contrast, differential interference contrast (DIC) and fluorescence microscopy, as described in the Methods section. Following CALG-V, a subset of washed cell samples was treated with the permeant chelator salicyl isonicotinoyl hydrazone (SIH) (50 µM) for 10 min (in order to reveal all Fe quenched complexes). Image analysis was carried out on confocal fluorescence images obtained after loading with CALG-Fe (fluorescence superimposed on phase contrast) or CALG-V (fluorescence) and on DIC images obtained after erythrophagocytosis. Results of cell image analysis of n = 3 independent experiments yielded statistically different (P < 0.05) parameters (mean values ± standard deviation determined in three areas of interest (one area per single cell) between WT and IS cells incubated with CALG-Fe: # of endosomes/cell: 22 ± 7 versus 56 ± 5; endosomes mean fluorescence (rel units): 36 ± 3 versus 28 ± 5; cell mean fluorescence (rel units): 11 ± 2 versus 19 ± 1; cell total fluorescence (rel units): 277 ± 143 versus 1096 ± 50. The mean numbers of erythrophagocytosed red blood cells (RBCs) per macrophage (WT vs. IS) were 1.2 ± 0.6 and 3.6 ± 1.8.