4.4.2. HOECHST Nuclear Staining

A549 cells in ibiTreat µ-slides 2 wells from ibidi (Martinsried, Germany) were incubated with H2O<sup>2</sup> + LPS with/without BUD, HPβCD, or BUD:HPβCD complex. Cells were then washed with PBS, covered with a 2000-fold dilution of HOECHST® 33,342 staining solution (Life technologies, Eugene, OR, USA) in PBS, and incubated 5 min at room temperature protected from light. Cells were then washed with PBS and imaged with a fluorescence microscope (λex/em = 350/461, DAPI filter set). Cells with bright and/or fragmented nuclei were considered apoptotic. The proportion of apoptotic cells was calculated from a total cell count of 400/well. H2O<sup>2</sup> + LPS concentrations were those preselected for LDH assay.
