Cellular distribution of tissue factor in fibroblasts. (A) Immunostaining of tissue factor. Nonpermeabilized and Triton X-100–permeabilized WI-38 lung fibroblasts were immunostained with polyclonal rabbit anti–human TF IgG (10 μg/mL), followed by Oregon Green–conjugated anti–rabbit IgG. Fluorescence was viewed with a Perkin Elmer UltraVIEW laser scanning confocal microscope. (B) Quantification of the plasma membrane and intracellular tissue factor. TF levels at the plasma membrane and in the intracellular pool were determined by measuring pixel density of the fluorescence of immunostained cells using 3-D reconstructed images of z slices. The difference in the pixel density between the nonpermeabilized and permeabilized cells was taken as an estimate of the TF in the intracellular compartment (n = 31 cells from 4 different experiments, mean ± SEM). (C) Functional activities of the cell surface and the intracellular tissue factor. Tissue factor activity at the cell surface was blocked by incubating the intact cells with anti-TF IgG (10 μg/mL) for 1 hour at 4°C. Unbound antibodies were removed and the cells were washed 3 times before they were permeabilized by Triton X-100 (0.01% for 10 minutes) or lysed by freeze-thawing. TF activity was measured by adding FVIIa (10 nM) and factor X (175 nM), and measuring the generation of FXa in a chromogenic assay. To demonstrate that there were no free/excess anti-TF antibodies in the experimental system, a small amount of purified relipidated TF (to permeabilized cells) or fibroblast cell lysate (to lysed cells) was added to cells whose surface TF activity was blocked by anti-TF antibodies or to a buffer (n = 3; mean ± SEM).