Third-generation BMECs were treated with different concentrations of hydrocortisone (HC; 0 mg·mL−1, 0.1 mg·mL−1, 0.2 mg·mL−1, 0.3 mg·mL−1, 0.4 mg·mL−1; Kingyork, China) for 24 h. The apoptosis of BMECs was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)/4′,6-diamidino-2-phenylindole (DAPI) double staining (Beyotime, China) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining (BD Biosciences).
Annexin 5 fluorescein isothiocyanate fitc propidium iodide double staining
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Sourced in China, United States
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining is a laboratory technique used to detect and quantify apoptosis (programmed cell death) in cell populations. Annexin V-FITC binds to phosphatidylserine, which is exposed on the surface of apoptotic cells, while propidium iodide (PI) stains the nuclei of cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. This dual staining method allows for the identification of early and late apoptotic cells, as well as viable and necrotic cells, using flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 fluorescein isothiocyanate fitc propidium iodide double staining
1
Hydrocortisone-Induced BMEC Apoptosis
2
Quantifying Neuronal Apoptosis via Annexin V-FITC/PI
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Annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) double staining (BD Bioscience, MA, USA) and flow cytometry were used to assess the effects of IL-6 on the induction of neuronal apoptosis. Neurons (1×106) from each group co-culture with IL-6 were harvested and washed twice with PBS. According to the manufacturer’s instructions, the cells were first incubated with 10 μL of Annexin V-FITC at 37°C for 15 min and subsequently counterstained with 5 μL of PI for 30 min in the dark. FITC/PI double staining was assessed by a BD FACSCalibur flow cytometer (BD Bioscience, MA, USA). Early apoptotic cells were defined as Annexin V-positive and PI-negative cells (quadrant A4), while late apoptotic or necrotic cells were defined as both Annexin V- and PI-positive cells (quadrant A2). Each sample was prepared in triplicate. Only Annexin V-positive cells were counted for statistical purposes.
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