Annexin 5 fluorescein isothiocyanate fitc propidium iodide

HLE cells obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), ghrelin (St Louis, MO, USA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT, Beyotime Institute of Biotechnology, Shanghai, China), Dulbecco's modified Eagle's medium (DMEM, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), Annexin V−fluorescein isothiocyanate (FITC)/propidium iodide (PI) (BD Biosciences, Mountain View, CA, USA), trypan blue (Zeye Institute of Biotechnology, Shanghai, China), acridine orange/ethidium bromide (AO/EB, Solarbio of Biotechnology, Beijing, China), anti-Bax, anti-Bcl-2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and hematoxylin–eosin (HE) kit (Jinqiao Biotechnology, Zhongshan, China).

In this experiment, a population of apoptotic and necrotic cells of the U87MG cell line was detected using an Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) detection kit according to the manufacturer’s protocol. Briefly, the tested U87MG cells were plated into 6-well culture dishes (2 × 10⁵ cells/well) for 24 h prior to the addition of hairy root extract of S. obtusifolia at the IC₅₀ concentration used in this study. Following 24 h incubation with both extracts, the percentage of apoptotic/necrotic cells was determined by the Annexin V–FITC/PI assay, according to our previous studies [60 (link)].

Cell cycle, cell apoptosis, and cell surface antigens were detected using flow cytometry. In brief, 48 h after treatment of KG-1, Kasumi-1, KG-1a, TF-1, or primary cells, the cell density was adjusted according to the cell viability assay and prepared for cell cycle analysis as described in our previous report [17 (link)]. Cells were stained with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA) to detect the apoptosis state in accordance with the manufacturer specifications. The CD34⁺/CD38⁻ population (Kasumi-1 cells) or CD34⁺/CD38⁻/CD96⁺ population (KG-1 cells) was obtained by incubating with CD34–FITC (Biolegend, San Diego, CA USA), CD38–R-phycoerythrin cyanine7 (CD38–PE-Cy™7; Biolegend), and CD96–R-phycoerythrin (CD96-PE; Biolegend) in Cell Staining Buffer (Biolegend); Annexin V–FITC and CD38–PE-Cy™7 were used for KG-1, KG-a, and TF-1 cells, and CD34–APC was also added for primary cells. The procedure was as follows: The cells (1 × 10⁶) were collected and resuspended in 100 μl Cell Staining Buffer, the antibody was added and incubated at 4 °C for 15 min, washed again with Cell Staining Buffer one time, and then subject to the apoptosis assay. To exclude non-viable cells, cells were stained with 7AAD before flow cytometry (BD Versa, San Diego, CA, USA) analysis for antigen detecting.

The apoptosis rate of HTR8/SVneo cells was done using flow cytometry and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (BD Biosciences, CA, USA) according to the manufacturer’s guidelines. In summary, after collecting HTR8/SVneo cells, the cells were incubated with 500 μL loading buffer, 5 μL Annexin V-FITC, and 10 μL PI solution as directed in Annexin-V-FITC Cell Apoptosis Detection Kit (Biovision, K101). The apoptosis rate was determined in a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was repeated three times, and the data were averaged.

Apoptosis was detected by staining with Annexin V-Fluorescein Isothiocyanate (FITC)/ Propidium Iodide (PI) (BD Biosciences) following the instructions of the manufacturer, then cells were analyzed by flow cytometry (FACScan, BD Biosciences), and the percentage of the cells in different phases was counted.