Quantifying Apoptosis and Necrosis in Cell Cultures

Assessment of apoptosis and necrosis in cultures was performed using DNA binding dyes—Hoechst 33342 and propidium iodide (PI). The analyses were performed under an Axiovert 200M confocal microscope with an LSM 5 PASCAL scanning head (Zeiss, Jena, Germany). Cell cultures for the analysis were carried out in a 35 × 10 mm tissue culture dish (SPL Life Sciences, Pocheon, South Korea) and inoculated with 2 mL of a cell suspension at a concentration of 1 × 10⁵ cells/mL. After 24 h incubation with the test fractions at a final concentration of 300 μg/mL and 2.5 mg/mL fibrinogen, 5 μL of Hoechst 33342 (0.4 mg/mL) and PI (0.5 mg/mL) mixed in a 2:3 ratio were added where applicable. The cultures were incubated for 5 min at 37 °C in the dark. After this time, the medium with the staining solution was removed and replaced with PBS buffer with Ca²⁺ and Mg²⁺ ions. The buffer exchange was followed by microscopic observations at an excitation wavelength of λ = 420 nm. The number of apoptotic and necrotic cells was calculated based on the observation of at least 1000 cells in randomly selected fields of view. Cells exhibiting blue fluorescence of fragmented nuclei were interpreted as apoptotic, whereas cells with pink fluorescence of whole nuclei were classified as necrotic. Each analysis was repeated three times [22 (link),35 (link)].