Detection of apoptosis by flow cytometry

Cells were seeded in cell culture medium in 24-well plates (Greiner Bio-One) overnight. Podocytes were seeded on six-well plates and grown at 33 °C, until they reached 70–80% confluency. Podocytes were differentiated for 10 days at 38 °C. All cells were treated with LCA for inidicated time, harvested using Accutase (Capricorne Scientific Ebsdorfergrund, Germany), washed twice with PBS (Gibco, Life Technologies, Darmstadt, Germany) and resuspend in Annexin V Binding buffer (10 mM Hepes, 140 mM NaCl and 0.25 mM CaCl₂). Apoptosis was detected by Annexin V-FITC (556419; BP Pharmingen, Heidelberg, Germany) and Propidium-Iodine (PI) (Invitrogen, Darmstadt, Germany) staining. Analysis was performed with a flow cytometer (FACSCanto™ II, Becton, Dickinson, and Company, Franklin Lakes, NJ, USA). Data was analyzed using BD FACSDiva™ (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA).