Telomere FISH Analysis of Adherent Cells

Telomere FISH was performed by using a PNA probe (Panagene) described as follows^{65 (link),66 (link)}. Peritoneal suspensions were collected and were adjusted to 1 × 10⁶ cells ml⁻¹ in DMEM. Then the cells were added to a six-well culture plates with glass slides and incubated at 37 °C for 2 h to allow adherence of macrophages to the glass slides surface. After the incubation, the supernatants containing nonadherent cells (mainly lymphocytes) were removed by gently washing three times with warm PBS. Adhered cells were swollen in KCl buffer (12.3 mM HEPES, 0.53 mM EGTA, 64.4 mM KCl), fixed in methanol/acetic acid (3:1), rehydrated in PBS, fixed in 4% formaldehyde and then dehydrated in a series of concentrations of ethanol. Slides were incubated with hybridization mixture (70% formamide, 10 mM NaHPO₄, pH 7.4, 10 mM NaCl, 20 mM Tris buffer, pH 7.5), placed on a 80 ℃ heating block for 5 min to denature chromosomal DNA and incubated with the PNA probe (0.05 mg ml⁻¹) for 2 h at room temperature. After washing, slides were mounted with Vectashield mounting medium containing DAPI (Vector Labs) and analyzed with a confocal microscope (Zeiss LSM 780).