Fitc annexin 5 apoptosis kit

The 1:1 proportion bone cement sample was chosen to be put into a centrifuge tube with 5 ml PBS in 37°C thermostatic box for releasing Nec-1 for 48 h. After that, the supernatant was collected to co-cultured with MC3T3-E1 cells, which is given by China Infrastructure of cell line resource. The in vitro experiment was divided into three groups, including control group, TNF-α group, and TNF-α plus sample releasing supernatant. MC3T3-E1 cells were cultured in 24-well plates at a density of 2 × 10⁵/ml with basal culture media (alpha Modified Eagle Medium, 15% fetal bovine serum, 100 IU penicillin-100μg/ml streptomycin, and 2.5μg/ml Fungizone) for 48 h. Then the medium was changed by fresh medium. After the cells grew to 80% confluence, the collected Nec-1 supernatant was added to co-culture with the cells for 1 h. Subsequently, the necroptosis was introduced by adding 10 μg/ml TNF-α for 48 h culturing. After that, three group cells were treated by trypsinization and then harvested by centrifuging at 3000 rpm for 5 min. Next, according to the manufacturer’s instructions, the cells were suspended and stained with Annexin V and PI by using a FITC Annexin V Apoptosis Kit (MultiSciences Biotech Co., Ltd, China). The experiment was repeated three times independently and the data were analyzed by FlowJo VX software.