Quantifying Podocyte Apoptosis

Following pharmacological treatment (48 h; Control, 30 µg/ml PAN, 100 µg/ml RTX, PAN + RTX), Hoechst 33342-staining of podocytes was performed as previously described^{25 (link)}. After collecting supernatant medium with detached cells, cultures were washed with PBS and detached using Trypsin–EDTA (Biochrom, Berlin, Germany) for 5 min at 37 °C, pelleted for 5 min at 1200 rpm, washed with PBS and resuspended in 2.5 ml growth medium. Hoechst 33342 (Sigma, München, Germany) was added to the medium to a final concentration of 1 µM/ml for 15 min at 37 °C. Cells were fixed for 15 min at room temperature with 4% formaldehyde (Fischar, Saarbrücken, Germany). After pelleting, cells were resuspended in 100 µl PBS, dispensed on a microscope slide and dried for 1 h in the dark. Slides were covered with ProLong Gold Antifade Reagent (Invitrogen, Karlsruhe, Germany) and coverslips. Images were randomly obtained by a Zeiss Axio Imager A1 fluorescence microscope and Axio Vision SE64 Rel. 4.9.1 software (Zeiss, Jena, Germany). Analysis was performed with ImageJ (https://imagej.nih.gov/ij/) software. Apoptosis was defined as percentage of cells with nuclear condensation/fragmentation. Condensed/fragmented nuclei were then counted by an observer unaware of the experimental conditions. For each sample in a given experiment, at least 200 randomly chosen cells were analyzed.