Aging Aging

74 Carmody et al.Table 1Summary of Assays and Probes Described in this Chapter aProbe/Assay Parameter measured Emission (nm) ChannelAnnexin-V PS translocation 515 FL-1TUNEL DNA fragmentation 515 FL-1Antigen analysis Antigen expression 580 FL-2PI DNA content 620 FL-2DHE Superoxide anion 605 FL-2DCFH/DA Peroxide 529 FL-1JC-1 Mitochondrial membrane 590 FL-2potentiala The table includes the emission peak of probes and the channel through which data shouldbe collected and analyzed.ruption of ∆ψ m is believed to occur through permeability transition (PT), aprocess that involves the opening of the mitochondrial PT pore, allowingrelease of solutes 1.5 kDa and smaller and subsequent disruption of ∆ψ m .Importantly, inhibitors of PT also inhibit apoptosis in several models ofapoptosis, supporting the view that disruption of mitochondrial function iscentral to the apoptotic process.The cell permeant fluorescent probe JC-1 can be employed to monitorchanges in ∆ψ m in intact cells. In the presence of a high ∆ψ m JC-1 forms whatare termed J-aggregates that fluoresce strongly at 590 nm (FL-2). Reduced∆ψ m results in a reduced FL-2 signal in JC-1-stained cells (Fig. 2A,B). Thismethod has been demonstrated to be quantitative in addition to qualitative andallows subpopulations of cells with different mitochondrial properties to beidentified (14).2. Materials2.1. Annexin V Assay1. Fluoresceinated Annexin V (Annexin V-FITC) (e.g., Bender MedSystems,Heidelberg, Germany). Protect from light and store at –20°C.2. Binding buffer: 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid](HEPES)/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 . Store at 4°C.3. PI. Protect from light and store at 4°C.4. Phosphate-buffered saline (PBS): 8.06 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , pH 7.4;2.27 mM KCl, and 137 mM NaCl.2.2. Terminal dUTP Nick End Labelling (TUNEL) Assay1. Fixation buffer: 2% (w/v) p-Formaldehyde in PBS, pH 7.4 (see Subheading 2.1.for PBS formulation).