Edu assay

We evaluated the galangin cytotoxicity using the Cell Counting Kit 8 (CCK8, Abcam) as previously described [19 (link)]. In brief, the cells were seeded into 96-well plates in the presence of various galangin concentrations for 24, 48, and 72 h. Ten μL of CCK-8 reagent was added to each well before incubation for two hours. The absorbance was measured at 450 nm. The EdU assay was performed following the manufacturer’s protocol (Abcam).

Cell proliferation was assessed using a Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) and EdU assay (Abcam, USA). Trophoblast cells were plated at 2×10³ cells/well in 96-well plates. Cell proliferation ability was determined at 24, 48, 72, or 96 hours using the CCK-8. At the above time point, the supernatant was removed, 10% CCK-8 solution was added to each well, and the cells were cultured for two more hours. The enzyme labeling instrument measured the absorbance at 450 nm wavelength. EdU experiments were carried out to analyze the proliferation of HTR-8 and BeWo cells according to the manufacturer’s instructions. The percentage of EdU positive cells (EdU positive/DAPI positive) was labeled as EdU positive cells. Five technical replicates were done per experiment, and three independent experiments were performed. Colony formation assay was performed as described. After transfection, cells were seeded to six-well plates at densities ranging from 100 to 40,000 cells per well and were put in an incubator for 10–14 days. After incubation at 37°C, cells were fixed with glutaraldehyde and stained with crystal violet. Colony formation for each condition was compared with that of untreated control cells. Results were obtained from five replicate wells, and each experiment was repeated three times.

EdU assay (Abcam; Cambridge, UK) was performed to evaluate the proliferation of T cells. T cells were treated with fixative solution and incubated for 15 min, then with permeabilization buffer and incubated for 15–20 min. The reaction mixture was added to fluorescently label EdU and incubated for 30 min. The cells were analyzed with flow cytometer (FACS CantoII flow cytometer) and FlowJo software (Becton Dickinson, San Jose, CA, USA), acquiring 10,000 events.