Annexin 5 fitc and propidium iodide detection kit

AFs were seeded in Ф35 mm confocal Petri dishes with a glass-bottom diameter of 20 mm at a density of 5 × 10⁴ cells/well, fixed in 4% paraformaldehyde (paraformaldehyde, phosphate and deionized water with a pH of 7.4) for 15 min, permeabilized in 0.1% Triton X-100 for 20 min, and then blocked with 0.5% goat serum in PBS. AFs were incubated with primary antibodies against light chain 3 (LC3), and p62 (Abcam, CA, USA) at 4 °C overnight and then incubated with secondary antibodies for 1 h. Finally, the nuclei were stained with flourished mounting medium with DAPI (Invitrogen, USA) intended for mounting slides and forming a semi-permanent seal for prolonged storage of slides at 4 °C for 10 min. Fluorescent images were captured by an Olympus IX51 microscope (Olympus, Tokyo). To investigate cell survival, AFs were washed with PBS and stained with Annexin V-FITC and propidium iodide (PI) detection kit (BD, USA). Briefly, the cells were resuspended in 100 μl binding buffer solution containing 5 μl Annexin V-FITC and 5 μl PI in the dark for 15 min and then analyzed by flow cytometry within 1 h after halting reaction. NRCMs and H9C2 cells stained with Annexin V, PI, or both were designated early apoptotic, necrotic, or late apoptotic cells. The flow cytometry data were analyzed using FlowJo V10 software.