Monocyte-derived macrophages show increased phagocytosis of E coli-GFP in the presence of SAA. (A) E coli-GFP association and (B) phagocytosis were measured by confocal microscopy and counting of bacteria associated with or internalized by macrophages counterstained with LysoTracker and Phalloidin-BODIPY. Data represent (A) percentage of GFP⁺ macrophages associated to bacteria and (B) phagocytic index, which refers to the number of bacteria internalized per 100 macrophages. Data represent mean ± SEM of quadruplicates from 4 individual donors. Statistical significance of samples with bacteria + SAA versus bacteria alone was calculated by Wilcoxon matched pair test (^*P < .05; ^**P < .01). (C-D) Binding and uptake or phagocytosis of E coli () or E coli in the presence of cytochalasin D (), or S pneumoniae (□) in the absence or presence of (C) purified SAA and (D) recombinant SAA (rSAA), was analyzed by FACS. Binding and uptake were expressed as the ratio of percentage of GFP⁺ macrophages in the presence of bacteria and SAA to that in the presence of bacteria alone. Increase in response to SAA was largely inhibited by the addition of cytochalasin D, implicating phagocytosis as the reason for increases in the presence of SAA. The dotted line represents unaltered bacterial association in the absence of SAA. Data represent mean ± SEM of 14 donors for E coli and 3 for S pneumoniae. Statistical significance of samples with bacteria + SAA versus bacteria alone was calculated from raw percentage of GFP⁺ cells by Wilcoxon matched pair test (^*P < .05; ^**P < .01 ^***P < .001).