Apoptosis assay by Hoechst 33342 staining

Hoechst 33342 is a blue, fluorescent dye that can penetrate the cell membrane and is used for apoptosis detection. Podocytes were plated into 24-well dishes (2×10⁵ cells/well) and were cultured under the aforementioned conditions. Then, cells were rinsed with PBS and incubated for 10 min at room temperature using 5 mg/ml Hoechst 33342. Staining was observed by fluorescence microscopy (Nikon Corporation) at ×400 magnification. The excitation wavelength was maintained at 380 nm. When apoptosis occurred, the nuclei appeared as dense or fragmented.