HRG mediates the binding of the copurified IgG to necrotic cells but not to viable cells. (A) Analysis of viable and necrotic (56°C for 30 minutes) Jurkat T cells by flow cytometry on the basis of Hoechst 33258 and annexin V–PE staining to determine viable, early apoptotic and necrotic cells. (B) Quantitative comparison of the ability of HRG in HRG^P or HRG^PID preparations (100 μg/mL) to bind to either viable or necrotic cells, as detected by flow cytometry. (C) Cellular localization of HRG^PID (100 μg/mL) bound to viable or necrotic cells as determined by CLSM. (D) Flow cytometric detection of IgG binding to viable and necrotic cells incubated with either HRG^P or HRG^PID preparations (100 μg/mL) in the absence of any additional IgG. Representative flow cytometric histograms are shown, with filled histograms representing Ab only control and open blue and red histograms representing IgG binding when HRG^P and HRG^PID preparations, respectively, were used. (E) Cellular localization by CLSM of HRG and IgG bound to necrotic cells after incubation with HRG^P (100 μg/mL). (F) Quantitative comparison of IgG^HRG (2 μg/mL) binding to necrotic cells either alone or in the presence of an approximately 1:1 molar ratio of HRG, with the HRG^P preparation being used as the source of HRG-IgG complexes. (G) Effect of increasing concentrations of HRG^PID (as molar ratio of HRG:IgG^HRG) on the binding of IgG^HRG to necrotic cells. IgG binding in panels F and G was determined by flow cytometry. Data in panels B, F, and G are expressed as fold binding above background mean fluorescence intensity (MFI), with error bars representing SEM (n = 3). *P < .05; **P < .01; ***P < .001.