Fluorescein dutp and terminal deoxynucleotidyltransferase

Apoptosis analysis was done as previously described [57 (link)]. Subconfluent PC3 and αT3-1 cells were plated on glass coverslips in 12 well plates under the standard culture conditions as described above. Twenty-four hr after the initial seeding, cells were serum-starved and then treated. At different times after treatment the cells were fixed with paraformaldehyde solution (4% in PBS (pH 7.4) for 1 h at 23 °C), washed with PBS and then incubated with 0.1% Triton X-100 in 0.1% sodium citrate (2 min, 4 °C), washed again with PBS, and incubated with terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) reaction mixture containing fluorescein-dUTP and terminal deoxynucleotidyltransferase (Roche Molecular Biochemicals, Germany) for 30 min at 37 °C. Preparations were analyzed by fluorescence microscopy.

To analyze apoptosis, sub-confluent PC3 and αT3-1 cells were plated on glass coverslips in 12 well plates under the standard culture conditions as described above. Twenty-four h after the initial seeding, cells were serum-starved and then treated. At different times after treatment the cells were fixed with paraformaldehyde solution (4% in PBS (pH 7.4) for 1 h at 15–25 °C), washed with PBS and then incubated with 0.1% Triton X- 100 in 0.1% sodium citrate (2 min, 4 °C), washed again with PBS, and incubated with terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) reaction mixture containing fluorescein-dUTP and terminal deoxynucleotidyltransferase (Roche Molecular Biochemicals Mannheim Germany) for 30 min at 37 °C. Preparations were analyzed by fluorescence microscopy.