The apoptosis of apoptotic cells was assessed by flow cytometry. 2 × 105 cells were seeded overnight in 6-well plates and then treated with IR for 24 h. Thereafter, the cells were collected, washed three times with cold PBS, stained with Annexin V-PE and 7-AAD (Vazyme, Nanjing, China) in binding buffer for 10 min at room temperature and in the dark and then quantified with a BD FACSCalibur flow cytometer (BD Sciences, USA).
Annexin 5 pe
Manufactured by Vazyme
Sourced in China
Annexin V-PE is a fluorescent-labeled protein that binds to phosphatidylserine (PS) on the surface of apoptotic cells. It is commonly used in flow cytometry and other cell-based assays to detect and quantify cells undergoing apoptosis.
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Lab products found in correlation
5 protocols using annexin 5 pe
1
Apoptosis Assessment by Flow Cytometry
2
Astrocyte Apoptosis Analysis Post H2O2 Exposure
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Astrocytes were incubated with or without 10 μg/ml of MP (Pfizer Inc., New York, NY, United States) after treatment with hydrogen peroxide (H2O2; 100 mM) for 24 h (Vieira et al., 2008 (link)). Cells were digested with trypsin without EDTA. Afterward, the incubation was terminated, and the cells were collected, centrifuged at 1,000 rpm at 4°C for 5 min, and the supernatant was discarded. The cells were washed twice with pre-cooled PBS, centrifuged at 1,000 rpm at 4°C for 5 min each time, and the supernatants were discarded. Cells were then resuspended and incubated in Annexin V-PE (Vazyme) and 7-AAD for 5 min at room temperature in the dark. The cells were then washed in PBS three times before being analyzed using a FACSVerse flow cytometer (BD Biosciences) running the FACSuite software. Data analysis was performed using the FlowJo software (Treestar).
3
Apoptosis Quantification by Flow Cytometry
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All cells were harvested and washed three times with phosphate-buffered saline (PBS), then incubated with Annexin V-PE (Vazyme) for 15 min at 37 °C in the dark. Subsequently, all cells were washed with FACS wash buffer (1 × PBS containing 0.5% BSA and 0.03% sodium azide), and apoptotic cells were analyzed via flow cytometry (BD Calibur).
4
Comprehensive Cellular Assays for Proliferation, Clonogenicity, Migration, and Apoptosis
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Cell proliferation assay: A total of 2 × 10^3 cells were seeded into 96-well plates and allowed to adhere for 24, 48, 72, and 96 h, respectively. Following this, 100 µL of medium containing 10% CCK8 was added into each well and incubated for a further 2 h. The optical density (OD) was then measured at 450 nm using a Microplate spectrophotometer. Colony formation assay: A total of 1 × 10^3 cells were seeded into 6-well plates and allowed to proliferate until visible clones emerged. The cells were then stained with crystal violet solution (Beyotime) and the number of colonies was counted macroscopically. Wound-Healing Assay: Transfected cells were seeded into 6-well plates and cultured until reaching greater than 80% confluence. A linear wound was subsequently created by a sterile pipette tip and the cells were incubated in serum-free medium for 24, 48, and 72 h, respectively. The migration of cells during healing was observed under an inverted microscope and quantified by measuring the wound width rate. Detection of apoptosis by flow cytometry: Harvested cells were washed with PBS and subsequently treated with 500 µl binding buffer, 5 µl Annexin V-PE, and 5 µl 7-AAD Staining Solution (Vazyme). Following a 15-minute incubation at room temperature in the dark, the samples were examined using a flow cytometry system.
5
Cell Cycle and Apoptosis Analysis
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The cell cycle distribution and apoptotic cell population were scrutinized as described previously (23 (link)). In brief, for the cell cycle, collected cells were fixed with 70% ethanol (Sangon Biotech, Shanghai, China) and stained with 0.5 ml propidium iodide (PI) (Beyotime Biotechnology, Shanghai, China) for 30 min. For cell apoptosis analysis, collected cells were stained with 5 μL Annexin V-PE and 5 μL 7-AAD for 10 min (Vazyme, Nanjing, China). The cells were examined using flow cytometry (BD, New Jersey, USA). The cell cycle data were analyzed with FCS Express Launcher software (De Novo Software), and the cell apoptosis results were analyzed with FlowJo software.
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