Fas and HLA-DR expression is lost during culture of TEC. Primary cultures of human TEC were established as described in Materials and Methods. TEC were collected after 2, 4, 7, or 10 days of primary culture by trypsin treatment and then labeled with FITC-conjugated anti–HLA-DR or anti-Fas antibodies. TEC were first incubated with anti-Fas monoclonal antibody for 30 minutes at 4°C, then washed in HBSS supplemented with 5% fetal serum calf, stained with biotin-coupled goat antimouse IgG antibody, washed, and incubated with streptavidin/Quantum Red conjugate. Cell labeling was analyzed on a FACScan flow cytometer. Vertical bars on Fas histograms indicate the Fas MFI level in TEC on day 2. Fas antigen expression fell during culture, similarly to HLA-DR antigen expression.