Apoptosis kits

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)].

Celastrol was supplied by Baoji Herbest Bio-Tech Co., Ltd. Media were provided by Invitrogen (America). Antisubstances of ATF4, phosphorylated eukaryotic initiation factor 2 alpha (P-EIF2α), EIF2α, STAT3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were supplied by Cell Signaling Technology (USA). Antisubstances, P-STAT3 and BCL2 Associated X (BAX), were provided by Abcam Co. (USA). Antibodies of B-cell lymphoma-2 (BCL-2) and secondary antibodies were purchased from Santa Cruz Biotechnology Inc. (USA). The Overall Protein Abstraction Kit was supplied by Boster Biological Technology (PRC). Dimethylsulfoxide (DMSO) and methylthiazolyldiphenyltetrazolium bromide (MTT) were provided by Sigma-Aldrich Co. (USA). Apoptosis kits were purchased from BD Pharmingen (USA). The Caspase 3 Assay Kit was purchased from Abcam Co. (Cambridge, MA, USA). ROS probes 2′,7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) and N-Acetyl-l-cysteine (NAC) were obtained from Beyotime Biotech (Nantong, China).