Apoptosis Assay for Microglial Cells

Annexin V/PI co-staining method was used to detect the degree of cell apoptosis. Human HMC3 microglial cells (1 × 10⁵/mL) were placed in 6-well plates and stimulated with LPS (1 μg/mL) for 24 h. The cells were then treated with TJZ-1 and TJZ-2, respectively, at concentrations of 1 μM and 10 μM, for 24 h. Celecoxib (10 μM) acted as an active control. Then, apoptosis assays were performed according to the protocol of the apoptosis kits (BD Biosciences, San Jose, CA, USA). Human HMC3 microglial cells were washed three times using serum-free medium, and 100 µL of binding buffer was added and dyed in the dark. Finally, a flow cytometer (Agilent NovoCyte, Palo Alto, CA, USA) was used for analysis at an excitation wavelength of 488 nm and an emission wavelength of 525 nm [39 (link)].