2.3. Hoechst 33342/PI Double Staining

Apoptosis and necrosis induced by exposure to mixtures and single particles were assessed by two assays: propidium iodide (PI) and Hoechst 33342 (Hoechst) staining, and annexin V/PI kit.

Hoechst/PI staining allowed us to distinguish viable, apoptotic, necrotic, and mitotic A549 cells after their exposure to NPs and mixtures. At least 300 cells per sample were counted under a fluorescence microscope (Zeiss-Axioplan, Carl Zeiss Microscopy GmbH, Jena, Germany) with a UV filter (365 nm), and scored as viable cells (H-positive and PI-negative cells, without special nuclear characteristics and with an intact plasma membrane), necrotic cells (PI-positive), apoptotic cells (bright H-positive cells with fragmented nuclei), and mitotic cells (H-positive with condensed chromosomes). Data are represented as the mean percentage ± standard error (SE) of the cell for each nuclear characteristic. The description of the annexin V/PI evaluation of apoptotic and necrotic cells by cytofluorimetric analysis is described in Section 2.6.