Annexin 5 apc 7 aad kit

Cardiomyocytes were dissociated from cell culture plates as described in “Generation of hiPSC-CMs”. Afterwards cells were washed with PBS, fixed with 1% formaldehyde solution (Merck) and permeabilized with perm/wash buffer for 10 minutes (1:10 dilution, BD Bioscienes cat# 554723). Then cells were incubated with different antibodies for 30 minutes at 4 °C in the dark. Finally antibody solution was washed out with PBS. Samples were measured immediately by FACS analysis, which was performed with CANTO II (BD). Compensation-measurement was considered if necessary by using OneCompeBeads (eBioscience). All antibodies were checked for correct concentration and non-specific fluorescence signal by previously testing with titration (1:10000 to 1:10 dilution) and comparison to negative control respectively by using isotype controls. The antibodies used for FACS analysis were AlexaFluor 647-conjugated cardiac Troponin T (1:10000 dilution, BD Biosciences cat# 565744), and PerCP/Cy5.5-conjugated CD 126 antibody (1:20 dilution, BioLegend cat# 352812). Apoptosis- and necrosis- ratio of LPS-treated hiPSC-CMs were measured with the Annexin-V-APC/7-AAD Kit (BioLegend cat# 640930, 420201).

The neuronal PC12 cells in the three groups were washed twice with pre-cooled PBS and processed using the Annexin V-APC/7AAD kit (BioLegend, San Diego, CA, USA) following the manufacturer’s instructions. Briefly, the cells were digested with EDTA-free trypsin (Solarbio, Beijing, China). After incubation with stain and binding buffer at room temperature for 15 min in darkness, the mixed cell suspension was transferred to flow tubes. The apoptosis rate was then determined using a flow cytometer (CytoFlex; Beckman Coulter, Inc., Brea, CA, USA).

For cell cycle analysis, cells were collected and fixed in 70% ethanol overnight. After being washed in phosphate-buffered saline (PBS) and digested with RNase A, cells were stained with propidium iodide (PI), followed by flow cytometric analysis. Cells apoptosis was detected using the Annexin V-APC/7-AAD kit (BioLegend, San Diego, CA, USA) following the manufacturer's instructions. The samples were measured on a FACSCanto (BD Biosciences, San Diego, CA, USA) flow cytometer and analyzed using the FlowJo 10.0 software (TreeStar, San Carlos, CA, USA).