Cells were seeded in triplicated in 24-well plates and treated with 2 μM MTX for 6 h. Then, the content was washed once and allowed to continue to culture in the drug-free medium for 48 h or 72 h. Subsequently, the cell samples were stained with annexin V-APC and PI (Elabsciences, Wuhan, China) and detected by flow cytometry (BD Biosciences, New Jersey, United States of America), the percentage of apoptotic cells was analyzed by FlowJo software (BD Bioscience).
Annexin 5 apc
Manufactured by Elabscience
Sourced in China
Annexin V-APC is a protein that binds to phosphatidylserine, a component of the cell membrane. It is commonly used in flow cytometry and fluorescence microscopy to detect and quantify apoptosis or programmed cell death.
Automatically generated - may contain errors
7 protocols using annexin 5 apc
1
Apoptosis Induction Assay with MTX
2
Lentiviral Transduction of hUC-MSCs for GFP Expression
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Lentivirus with green fluorescent protein (GFP) was purchased from (GKN, China, product No.: LVCON313). The 3rd generation hUC-MSCs were seeded at a density of 1 × 105/well and incubated in 6-well plates 18–24 hours before transfection. The next day, lentiviruses with GFP fluorescent markers were transfected. The number of cells at the time of transfection was approximately 3×105/well. The multiplicity of infection (MOI) was 20. The medium was changed after 24 hours of co-culture. Fluorescence was observed with an inverted fluorescence microscope. Based on the fluorescence intensity, cells that failed to stably express GFP were screened with 4 μg/mL of puromycin. After the stable expression of GFP was established, we examined whether cell proliferation and apoptosis were affected before and after transfection using a cell counting kit-8 (CCK-8) and apoptosis kits (Annexin V-APC and 7-AAD, Elabscience, China). Ultra-low adsorption round-bottom 96-well culture plates (Corning 7007, USA) were sterilized with UV light prior to the spheroidization of hUC-MSCs. Then, 100 μL of hUC-MSCs suspension at a concentration of 2.5×106/mL was added to each well and the cells were gently shaken to concentrate them at the bottom of the plate. After 48 hours of incubation, hUC-MSCs spheres were formed and removed from the wells with a pipette.
3
Cell Cycle and Apoptosis Analysis
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Cells were seeded into six-well plates at 2 × 105 cells per well and were treated as indicated. For the cell cycle assay, after collection, the cells were washed with cold phosphate-buffered saline (PBS), fixed with 70% cold ethanol, and stored at −4 °C overnight. Before analysis, the cells were stained with propidium iodide (PI) and RNase A (BD Biosciences, Franklin Lakes, NJ, USA). For the apoptosis analysis, after collecting, cells were either stained with PI and Annexin V–FITC (BD Biosciences) or Annexin V–APC (Elabscience, Wuhan, China). Both the cell cycle and apoptosis assays were assessed using a flow cytometer (BD Biosciences).
4
Apoptosis Detection by Flow Cytometry
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After cell transfection, 106 cells from each group were washed and stained Annexin V-APC and 7AAD (Elabscience, Wu Han, China) at room temperature for 15 min. Flow cytometry (BD FACSCalibur, New Jersey, USA) was used to evaluate the proportion of apoptotic cells (low right corner of the flow cytometry graph regarded as apoptotic cells).
5
Annexin V-APC and PI Apoptosis Assay
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Cells with different treatments were collected, washed by PBS and resuspended. About 5 × 105 suspended cells were centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were suspended with 100 μL diluted 1 × Annexin V Binding Buffer, and 2.5 μL Annexin V-APC and 2.5 μL PI staining solution (Elabscience, China) were added to cell suspension for incubating at room temperature away from light for 15–20 min. Then, 400 μL diluted 1 × Annexin V Binding Buffer was added, and the samples were mixed and detected by flow cytometry (Thermo, USA) [19 (link)].
6
Annexin V-APC and 7-ADD Apoptosis Assay
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The cells were resuspended in 1× annexin V binding working solution and then stained with annexin V-APC and 7-ADD (Elabscience, Wuhan, China) for 30 min at room temperature. A flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA, USA) was used. The apoptosis assay was performed 48 h after 8-Gy irradiation.
7
Apoptosis and Cell Cycle Analysis
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Cells were cultured in 6-well plates (2×105 cells/well) and collected after treatment. For apoptosis analysis, 5 μL Annexin V–APC (Elabscience, Wuhan, China) or Annexin V–FITC (BD Biosciences) and propidium iodide (PI) were added to the cells using binding buffer for staining, with the detection performed after 20 min of darkness. For cell cycle analysis, cells were fixed in pre-cooled ethanol for 1 h. Before analysis, the cells were stained with PI and RNase A (BD Biosciences, Franklin Lakes, NJ, USA). Cell cycle and apoptosis assays were performed using a flow cytometer (Beckman, USA).
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