2.9. Annexin V/propidium iodide analysis

One day prior to the experiment, the cells were seeded in 6-well plates (Corning) such that on the treatment day 75% confluence was reached. In particular, the following cell numbers were seeded: 4 × 10⁵ (U2OS, A2780), 6 × 10⁵ (HaCaT), 2 × 10⁵ (RPE-1), 3.3 × 10⁵ (VH10), 7 × 10⁵ (Podocytes). Regarding the PBMCs, 2 × 10⁶ cells were used for the treatment after the isolation from whole blood. The cells were treated as indicated in the respective figure. 24 h after the treatment, medium was removed and cells were washed with PBS. Cells were detached with trypsin and the cell suspension was added to the collected medium and PBS. After centrifugation at 300 ×g for 10 min, the pellet was resuspended in 5 ml ice-cold PBS and the cell number was determined by using a CASY cell counter. The volume containing 2 × 10⁵ cells was transferred into a new reaction tube and the cells were pelleted at 4 °C. Subsequently, the cells were resuspended in 100 µl Annexin V binding buffer (10 mM HEPES, pH 7.4; 140 mM NaCl; 2.5 mM CaCl₂) and stained with one drop of the Annexin V APC ready flow conjugate (Thermo Fischer Scientific) as well as 5 µl propidium iodide (PI) (1 mg/ml; Sigma-Aldrich). After incubation of the cells in the staining solution for 15 min in the dark, 400 µl Annexin V binding buffer were added and the samples were kept on ice until analysis with a FACSLyric instrument (BD Sciences). 10,000 events per condition were analyzed. The amount of viable, apoptotic, and dead cells was calculated using the FlowJo software (version 10.7.1; Becton Dickinson & Company).