3.6. Determination of Apoptotic and Necrotic Cell Fractions by Fluorescence Microscopy (Double Staining of Cells with Fluorescent Dyes Hoechst 33258 and Propidium Iodide)

The simultaneous use of two fluorescent dyes (propidium iodide and Hoechst 33258; Sigma, St. Louis, MO, USA), whose mechanism of penetration into the cell and the fluorescence spectrum are different, allows the identification of four types of cells in the same sample: live, early apoptotic, late apoptotic, and necrotic. Propidium iodide (PI) has a negative charge and only penetrates cells with damaged cell membranes, which allows the identification of necrotic cells or those present in late phases of apoptosis. Hoechst 33258 freely penetrates through the intact membrane of living and early apoptotic cells, thus enabling their identification. As a result of the dye penetration through intact biological membranes, it stains the DNA of the cell nucleus a light blue color.

Both fluorochromes are excited by ultraviolet light—propidium iodide has orange-red fluorescence, while Hoechst 33258 has blue fluorescence. Staining of cells with a mixture of both dyes allows one to distinguish four fractions of cells differing in fluorescence in the microscopic image:

Cells were seeded in 12-well plates in appropriate culture medium 24 h prior to experimentation. After this time, the analyzed compounds were added to the cells at the determined IC₅₀ concentration. Cells were incubated with compounds for 24 h. At the end of incubation, the medium from each well was removed and 3 mL of HBSS containing Hoechst 33258 (0.13 mM) and PI (0.23 mM) were added. The cells were incubated with the fluorochromes for 5 min at room temperature, in the dark. After this time, HBSS with fluorochromes was removed and fresh HBSS was added to the cells. The analysis was performed with a fluorescence microscope (Olympus IX70, Japan) under 400× magnification. Cells were classified as live, apoptotic, or necrotic on the basis of their morphological and staining characteristics.