Annexin 5 and pi apoptosis detection kit

For Annexin V and PI staining, BMDCs were infected with MAH (MOI of 1) in the presence or absence of LPS (100 ng/ml) at 37°C. After 24 h, the cells were harvested, washed twice with PBS, and stained using Annexin V and PI apoptosis Detection kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s instructions. The cells were analyzed using a LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA). Under the same culture conditions for Annexin V and PI staining, Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Tabaru, Japan) was added according to the manufacturer’s instructions to each well for 1 h at 37°C. Viable cells were analyzed by the absorbance at 450 nm, using a microplate ELISA reader.

To measure the effect of compounds on apoptosis and cell cycle, cells were incubated with the agents or DMSO for 24 h and then washed by cold PBS. For measure apoptosis, cells (10 ⁶) were stained with Annexin V and PI apoptosis detection Kit (BD Biosciences, Franklin lakes, NJ. Cat#556570), following the kit guidelines and analyzed by flow cytometer. For cell cycle analysis, drug treated and control cells (10⁶) were first fixed by cold 75% ethanol overnight at − 20 °C, washed once with cold PBS, stained in PI for 40 mins at 37 °C and then analyzed by flow cytometer, as described above.