Fluorescein diacetate (FDA)-propidium iodide (PI) double dye analysis to determine the influence of SAEW on cell activity

The influence of SAEW on cell activity was detected by FDA (West Asia reagent, China) and PI (Blue skies biotechnology, China). FDA is a non-polar, hydrophobic, non-fluorescent esterified compound, which readily permeates the cell membrane and is hydrolyzed by non-specific esterases producing a fluorescein. FDA was used to indicate the presence of active esterase. Meanwhile, PI was utilized to assess cell membrane integrity. Cells with an intact cell membrane and inactive esterase cannot be stained with FDA or PI. After treatment with SAEW (PBS as a blank control) for pre-set times, samples were centrifuged at 5000 rpm for 5 minutes in a refrigerated centrifuge and washed once. Then, the samples were divided into two groups: those that were dyed immediately and those that were dyed after a 4-h incubation. In the immediately dyed group, cells were resuspended in one hundred microlitres of binding buffer. Ten microlitres of FDA working solution and 2.5 μL of PI were added to each sample, which were then incubated in the dark for 10 minutes. In the group that was dyed after the 4-h incubation, cells were resuspended in 1 mL of sterile broth and incubated for 4 h. Then, the samples were centrifuged at 5000 rpm for 5 minutes in a refrigerated centrifuge and washed once before the staining step. Approximately 10⁶ cells were collected for each sample. The treated cells were detected with a BD flow cytometer.