Cells were plated in 6 well culture plates and incubated overnight. Treatments with helichrysetin were performed, 4.5, 9, and 18 μg/mL and from 6 to 72 h. After treatment, cells were harvested and washed twice with PBS, and staining was then performed according to assay kit's manufacturer's instructions. Annexin-V/fluorescein isothiocyanate (FITC) assay (BD Biosciences), Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Invitrogen, US) and JC-1 mitochondrial membrane potential (MMP) assay (BD Biosciences) were performed to analyze cell membrane integrity, internucleosomal DNA fragmentation, and mitochondrial membrane, respectively. For cell cycle analysis, cells were stained with propidium iodide (PI). All cells were acquired using Accuri C6 flow cytometer by capturing 10,000 events per run (BD Biosciences).
Annexin 5 fluorescein isothiocyanate fitc assay
Manufactured by BD
Sourced in United States
The Annexin-V/fluorescein isothiocyanate (FITC) assay is a laboratory technique used to detect and quantify apoptosis, or programmed cell death. Annexin-V is a protein that binds to phosphatidylserine, which is exposed on the surface of cells undergoing apoptosis. FITC is a fluorescent label that is conjugated to Annexin-V, allowing for the visualization and measurement of apoptotic cells through flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 fluorescein isothiocyanate fitc assay
1
Helichrysetin-Induced Apoptosis Evaluation
2
Apoptosis and ROS Assay in HepG2 Cells
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After stimulation, HepG2 cells were collected, a flow-based Annexin V/fluorescein isothiocyanate (FITC) assay (BD) was used to measure the apoptosis of HepG2 cells by following the manufacturer’s instructions. The cells in FITC-positive fraction were considered apoptotic. For ROS, 2 × 105 HepG2 cells were seeded in 12-well plates. After stimulation, dichloro-dihydro-fluorescein diacetate (Applygen Technologies, Beijing, China) were used to incubated cells at 37 °C for 30 min in the dark. Analyses were performed with a FACScan-420 flow cytometry instrument (Becton-Dickinson, USA).
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