Figure 6. Expression of Bcl-x Isoformsin O3 T Cell Clone Stimulated by Plate-Bound Anti-CD3 Antibody for 5 hr and Sorted by Flow Cytometry.

(Left) After stimulation of the O3 T cell clone by plate-bound anti-CD3 antibody for 5 hr, cells were subjected to staining with Hoechst 33342 dye and PI. After dead cells were gated out, activated cells were sorted into two subpopulations by flow cytometry, Hoechst-high (apoptotic) and Hoechst-low. Total RNA from both Hoechst-positive and Hoechst-negative subpopulations was extracted as described in Experimental Procedures. RT-PCR amplification of IFNγ fragment and IL-2 fragment after stimulation by anti-CD3 in both Hoechst-high and Hoechst-low cells confirmed activation of O3. Bcl-xγ was selectively expressed in Hoechst-low cells but not in Hoechst-high (apoptotic) cells, while all other Bcl-x isoforms are expressed in both forms. Failure to detect Bcl-xγ in Hoechst-high cells did not result from degraded preparations of total RNA or cDNA, since β-actin and other Bcl-x isoforms were detected in these samples.

(Right) The PCR-amplified fragments analyzed on agarose gel were scanned and quantitated using an IS-1000 digital imaging system, followed by normalization, as described above.