Figure 2.

Isolation of Fas-negative and Fas-positive cholangiocarcinoma populations. A1: Fas expression of cholangiocarcinoma cells before flow cytometric sorting. Cells were incubated with PE-conjugated anti-human Fas monoclonal antibody at 4°C for 30 minutes and washed with FACS butter (PBS, 5% FCS). The surface Fas antigen of cells was measured by flow cytometry compared with the control incubated with PE-conjugated anti-mouse IgG. A2: Isolation of Fas-positive and Fas-negative subsets by flow cytometric sorting. Cells were labeled as described above and sorted by flow cytometry into cells (10%) at the far left side of PE fluorescence peak (Fas-negative) and cells (10%) at the far right side of PE fluorescence peak (Fas-positive). These cells were incubated in RPMI 1640 complete medium for 1 week. Fas antibody (0.1 μg/ml) was added in the medium of the Fas-negative cells. Fas expression of Fas-negative (solid line) and Fas-positive (dotted line) were measured by flow cytometry. A3: Cloned Fas-negative and Fas-positive cell lines. The sorted Fas-negative and sorted Fas-positive cells were diluted and cells grown from a single cell were subsequently transferred onto tissue culture plates to generate cloned cells. The figure shows Fas expression of a Fas-negative clone (solid line) and a Fas-positive clone (dotted line). Fluorescence intensity is plotted on the x-axis; cell counts on the y-axis. B: Fas mRNA determined by RT-PCR. The mRNAs of sorted and cloned Fas-negative and Fas-positive cells (as indicated) were prepared using RNAzon kit and RT-PCR of Fas in sorted (lane 1) and cloned Fas-negative cells (lanes 2–3), as well as sorted (lane 4) and cloned Fas-positive cells (lanes 5–6) were performed. Fas was amplified using the primers of Fas (500 bp, see Materials and Methods). The 500-bp Fas DNA band is marked.