Fig. 3. Time (A) and concentration (B) dependence of native and tyrosylated Oregon-HDL₃ cell association and fluorescent staining of cells illustrating Oregon HDL₃ cell association (C). J774-A1 were cholesterol-loaded with AcLDL (50 µg apoB/ml) as described in Materials and Methods. After an 18 hour equilibration period, cells were incubated (37°C) for different times with 25 µg apo A-I/ml of native (•) or tyrosylated () Oregon-HDL₃ (A), or with different concentrations of native (•) or tyrosylated () Oregon-HDL₃ for 3 hours (B). These assays were performed in the absence or presence of a 50-fold excess of the corresponding unlabeled HDL₃. Cells were then extensively washed with DMEM-5% LPDS and PBS, and cell-associated fluorescence was measured by flow cytometry. Specific Oregon-HDL₃ binding was determined by subtracting the measurements made in the presence of 50-fold excess from the corresponding unlabeled HDL₃. The values are expressed as the means±s.e.m. of four independent experiments using different lipoprotein preparations. C shows the fluorescence microscopy of J774-A1 cells at 3 and 6 hours following Oregon-HDL₃ uptake (100 µg apo A-I/ml). ^*P<0.003.