Electron microscopy, annexin V labeling, and DAPI staining were performed as described previously (Madeo et al., 1997 (
link)). For the TdT-mediated dUTP nick end labeling (TUNEL) test, cells were prepared as described (Madeo et al., 1997 (
link)), and the DNA ends were labeled using the In Situ Cell Death Detection Kit, POD (
Boehringer Mannheim). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (
link)). The slides were rinsed with PBS and incubated with 0.3% H
2O
2 in methanol for 30 min at room temperature to block endogenous peroxidases. The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100 and 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (terminal deoxynucleotidyl transferase 200 U/ml, FITC-labeled dUTP 10 mM, 25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride;
Boehringer Mannheim) for 60 min at 37°C, and then rinsed 3× with PBS. For the detection of peroxidase, cells were incubated with 10 μl Converter-POD (anti-FITC antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 min at 37°C, rinsed 3× with PBS, and then stained with DAB-substrate solution (
Boehringer Mannheim) for 10 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck). As staining intensity varies, only samples from the same slide were compared.
Free intracellular radicals were detected with dihydrorhodamine 123, dichlorodihydrofluorescein diacetate (dichlorofluorescin diacetate), or dihydroethidium (hydroethidine;
Sigma Chemical Co.). Dihydrorhodamine 123 was added ad-5 μg per ml of cell culture from a 2.5-mg/ml stock solution in ethanol and cells were viewed without further processing through a rhodamine optical filter after a 2-h incubation. Dichlorodihydrofluorescein diacetate was added ad-10 μg per ml of cell culture from a 2.5 mg/ml stock solution in ethanol and cells were viewed through a fluorescein optical filter after a 2-h incubation. Dihydroethidium was added ad-5 μg per ml of cell culture from a 5 mg/ml aqueous stock solution and cells were viewed through a rhodamine optical filter after a 10-min incubation. For flow cytometric analysis, cells were incubated with dihydrorhodamine 123 for 2 h and analyzed using a FACS
® Calibur (
Becton Dickinson) at low flow rate with excitation and emission settings of 488 and 525–550 nm (filter FL1), respectively.
Free spin trap reagents
N-tert-butyl-α−phenylnitrone (PBN;
Sigma-Aldrich) and 3,3,5,5,-tetramethyl-pyrroline
N-oxide (TMPO;
Sigma-Aldrich) were added directly to the cell cultures as 10-mg/ml aqueous stock solutions. Viability was determined as the portion of cell growing to visible colonies within 3 d.
To determine frequencies of morphological phenotypes (TUNEL, Annexin V, DAPI, dihydrorhodamine 123), at least 300 cells of three independent experiments were evaluated.
Madeo F., Fröhlich E., Ligr M., Grey M., Sigrist S.J., Wolf D.H, & Fröhlich K.U. (1999). Oxygen Stress: A Regulator of Apoptosis in Yeast. The Journal of Cell Biology, 145(4), 757-767.
