CD163 interaction and endocytosis of peroxide-treated and untreated Hb and Hb-Hp. (A) CD163-HEK cells were incubated with 20 μg/mL native or H₂O₂-treated Hb or Hb-Hp. After 30 minutes, cells were washed, fixed, and stained with an anti-Hb antibody (red fluorescence, Alexa 568). Nuclei were stained with DAPI (blue). Images were taken with a Carl Zeiss epifluorescence Axioskope-2 (20×/0.43 objective) equipped with an AxioCam-MR digital camera and Axio-Vision software. Results of digital image analysis and quantification of red fluorescence (per cell) that indicates cellular Hb uptake are shown in the graph. Bars represent mean plus or minus SD of 3 independent experiments performed in triplicates with at least 10 images quantified per coverslip. (B) Equal binding of nonoxidized Hb-Hp (top) and oxidized Hb-Hp (bottom) complexes was confirmed by Biacore analysis over a range of concentrations (from 2.5-200 nM) normalized to Hb. (C) CD163-HEK cells were incubated with 3 μg/mL fluorescent Hb-Hp₆₃₃ in the presence or absence of different concentrations of nonfluorescent competitors (● = Hb-Hp [1-1]; ○ = oxidized Hb-Hp [1-1]). After 30 minutes, cells were washed, trypsinized, and fluorescence was determined by fluorescence-activated cell sorting (FACS). Mean channel fluorescence values are given as percentage of the Hb-Hp₆₃₃ fluorescence in the absence of any competitor. (Mean ± SD from 3 independent experiments.)