Transdetect annexin 5 fitc pi cell apoptosis detection kit

H. pylori cells were grown as described in the ‘Cell culture’ section above. Briefly, H. pylori cells were scraped from the Mueller-Hinton agar plates with or without daphnetin. To monitor the degree of cell membrane structural changes [17 (link)], a TransDetect Annexin V-FITC/PI Cell Apoptosis Detection Kit (Transgen Biotech, Beijing, China) was used. For membrane depolarization experiment [17 (link)], staining of cells were performed using DiBAC (Invitrogen, Waltham, MA, USA). To determine the integrity of cell membrane, a bicinchoninic acid (BCA) protein assay kit was used. Briefly, H. pylori cells (0.5 McFarland) were cultured with or without daphnetin in Brucella broth for 24 h. The samples were centrifuged at 4 °C, the supernatants were treated with BCA assay reagent, and OD at 595 nm was measured [43 ].

Cell apoptosis was measured by TransDetect^® Annexin V-FITC/PI Cell Apoptosis Detection Kit (Transgen, FA101, China). After treatment with various concentrations of IFN-λ1 for 24 h and infected by S. aureus, keratinocytes (2 × 10⁵ cells) were harvested, resuspended in 100 μl one x Annexin V binding buffer and incubated with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) for 15 min in the dark at room temperature. The samples were detected by a Beckman Coulter FC500 cytometer immediately, and the FlowJo software version 10.4 (BD, United States) was used for data analysis.

The effects of GSPs on apoptosis in HepG2 cells was analyzed using flow cytometry according to the instructions of TransDetect Annexin V-FITC/PI Cell Apoptosis Detection Kit (Transgen Biotech, Beijing, China). Briefly, cells were seeded in 6-well plates and cultured for 24 h in a humidified incubator containing 5% CO₂ at 37 °C. After cells were treated with 10 mg/L of GSPs for 24 or 48 h, adherent cells collected by trypsinization and floating cells were collected by centrifugation. These collected cells were washed twice with cold PBS and resuspended using 100 μL of ice-cold 1 × Annexin V Binding buffer followed by a mix of 5 μL of Annexin V-FITC and 5 μL of PI. The cells were then incubated in the dark at room temperature (20–25 °C) for 15 min before 400 μL of ice-cold 1 × Annexin V Binding buffer was added. Finally, the stained cells were detected using a FACSCalibur flow cytometer (BD biosciences, San Jose, CA, USA), and the data were analyzed by the Cell Quest Software (BD Biosciences).