Annexin 5 standard binding buffer

Supernatants were collected from cells and were assessed using the CytoTox96® non-radioactive cytotoxicity assay (Promega) at a wavelength of 490 nm. Percentage of LDH release was calculated as 100 × (experimental LDH-spontaneous LDH)/(maximum LDH release-spontaneous LDH). Cell death was also detected by propidium iodide permeabilization. For flow cytometry, cells were collected using accutase (Capricorn Scientific, Ebsdorfergrund, Germany), washed with cold Dulbecco’s PBS (DPBS) and centrifuged at 500 g and 4 °C for 10 min. Subsequently, cells were resuspended in a 100-µl annexin binding buffer containing 5 µl annexin V-FITC and 2.5 µl 7-aminoactinomycin D (7-AAD; BioLegend, Koblenz, Germany). Fluorescence-activated cell sorting (FACS) analyses were performed in the Annexin V standard binding buffer (BioLegend, San Diego, CA, USA) containing 10 mM Hepes, 140 mM NaCl and 2.5 mM CaCl. For each measurement, at least 10,000 cells were analyzed by flow cytometry at 37 °C (BD AccuriTM C6, St. Ives, UK). 7-AAD, a non-permeant dye, was used to identify cells with plasma membrane leakage. Freshly isolated macrophages were stained with propidium iodide staining to detect RSL3/erastin-induced cell death.

Cells were collected using accutase (Capricorn Scientific, Ebsdorfergrund, Germany), washed with cold Dulbecco’s PBS (DPBS) and centrifuged at 500 g and 4 °C for 10 min. Subsequently, cells were resuspended in 100 μL annexin binding buffer containing 5 μL annexin V-FITC and 2.5 μL 7-aminoactinomycin D (7-AAD; BioLegend, Koblenz, Germany). Cells were incubated with 1 μM ionomycin for 10 min or erastin (5 µM)/RSL3 (0.1 µM) (6 h), or melittin (0.5 µM; 14 h), respectively. Reactions were stopped by adding 400 μL DPBS and cells were analysed immediately. Fluorescence activated cell sorting (FACS) analyses was performed in Annexin V standard binding buffer (BioLegend, San Diego, CA, USA) containing 10 mM Hepes, 140 mM NaCl and 2.5 mM CaCl. For each measurement, at least 10,000 cells were analysed by flow cytometry at 37 °C (BD AccuriTM C6, St. Ives, UK) 7-AAD, a non-permeant dye, was used to identify cells with plasma membrane leakage. Freshly isolated macrophages were stained with propidium iodide staining to detect RSL3/erastin-induced cell death.