Quantitative Telomere Length Analysis

Q-FISH was performed as we previously described [49 (link)]. Briefly, all cells were maintained in the indicated conditions. On the harvesting day, 0.1 µg/mL colcemid (Invitrogen) was added for 6 h. Cells were collected by Accutase, incubated with 0.075 M KCl, fixed in cold methanol/acetic acid (3:1) and stored at 4 °C. Metaphase spreads were prepared as previously described [50 (link),51 (link)], and telomere FISH was performed using a red fluorescence-conjugated Telomere PNA probe (AF546-OO-CCCTAACCCTAACCCTAA) (IDT). Chromosomes were stained with 0.5 μg/mL DAPI. For quantification and fluorescence intensity of telomere size, metaphase spreads and telomeres were captured by Nikon CSU-W1 Spinning Disk Confocal microscope and analyzed by TFL-TELO [28 (link)].