Quantification of apoptosis was performed by cell cycle analysis. Cells were harvested by trypsinization and lysed in hypotonic buffer. Isolated nuclei were stained for 1 h with 40 mg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). Cells in G1, G2, and S-phase, as well as sub-G1 cells, were quantified by flow cytometry at FL3A using a FACS Calibur (BD Bioscience, Bedford, MA, USA). Due to the washing out of small DNA fragments, nuclei with less DNA than G1 (sub-G1) correspond to apoptotic cells.
Cytotoxicity was determined in cell culture supernatants by quantification of released lactate dehydrogenase (LDH) activity. Cells were stained by a cytotoxicity detection assay (Roche Diagnostics, Penzberg, Germany) and analyzed by an ELISA reader. Triton x100-treated cells (0.7%) were used as a positive control.
Cell viability was determined by staining cells with calcein-AM (PromoCell, Heidelberg, Germany; AM = acetoxymethylester), which is converted in viable cells through intracellular esterase activity to green-fluorescent calcein. Cells, grown and treated in 24-well plates, were harvested by trypsinization and stained for 1 h with 2.5 µg/mL calcein-AM at 37 °C. After labeling, cells were washed with PBS and measured by flow cytometry (FL2H).
Cytotoxicity was determined in cell culture supernatants by quantification of released lactate dehydrogenase (LDH) activity. Cells were stained by a cytotoxicity detection assay (Roche Diagnostics, Penzberg, Germany) and analyzed by an ELISA reader. Triton x100-treated cells (0.7%) were used as a positive control.
Cell viability was determined by staining cells with calcein-AM (PromoCell, Heidelberg, Germany; AM = acetoxymethylester), which is converted in viable cells through intracellular esterase activity to green-fluorescent calcein. Cells, grown and treated in 24-well plates, were harvested by trypsinization and stained for 1 h with 2.5 µg/mL calcein-AM at 37 °C. After labeling, cells were washed with PBS and measured by flow cytometry (FL2H).