Telomere length analysis by automated flow-FISH of CD4⁺ T-cell clones transduced with hTERT. Naive CD4⁺ T-cell clones were transduced with hTERT and a GFP control vector. After transduction cells were sorted for GFP expression and further expanded. Telomere length was measured by automated flow-FISH. (A) Telomere fluorescence histograms at different PDs. Once the average telomere length reached approximately 3.3 kilobase (kb), a population of cells with double the telomere signal was observed. Interestingly, this population also increased in size on further culture. (B) Cell-cycle analysis also revealed an increasing fraction of cells with 4N DNA with further increase of PDs. The cells were sorted and cytospins were prepared. DNA was stained with DAPI and analyzed with a Zeiss Axioplan II fluorescence microscope. Chromatin bridges between nuclei were observed, suggesting that cells failed to complete anaphase. (C) To analyze live cells, samples were also stained with Hoechst 33342. Cells were sorted for their DNA content, cytospins were prepared and stained with Wright-Giemsa stain. Again, binucleated cells with chromatin bridges between nuclei were observed.