The apoptotic studies were carried out using Guava Nexin Reagent (Millipore). Cells with different types such as normal cells, early apoptotic cells and late apoptotic cells can be distinguished by this apoptosis study using flow cytometer60 (link). The Guava Nexin assay uses two stains (annexin V and 7-amino actinomycin D (7-AAD)) to quantitate the percentage of apoptotic cells. It was conducted according to the manufacturer’s protocol. In brief, after treatment with the alloys extracts for 24h, U2OS cells were collected and resuspended in 100ml medium supplemented with 100 μl 1% FBS. The cells were then incubated with 100 μl Annexin V-PE and 7-AAD labeling solution (Millipore) for 20 min at room temperature in the dark. Cells positive for AnV only (early apoptotic) or AnV and 7-AAD-positive cells (late apoptotic) were quantified by Guava EasyCyte 5HT flow cytometer. The data were analyzed by Guava Nexin Software v2.2.2.
Annexin 5 pe and 7 aad labeling solution
Manufactured by Merck Group
Annexin V-PE and 7-AAD labeling solution is a laboratory reagent used for the detection and analysis of apoptosis, a type of programmed cell death. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which becomes exposed on the cell surface during apoptosis. 7-AAD is a fluorescent dye that binds to DNA, allowing the identification of cells with compromised cell membranes, such as late apoptotic or necrotic cells. This solution provides a method for the simultaneous detection and quantification of apoptotic and necrotic cells using flow cytometry or fluorescence microscopy.
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2 protocols using annexin 5 pe and 7 aad labeling solution
1
Quantifying Apoptosis in U2OS Cells
2
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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Cell cycle conditions were determined using propidium iodide (PI, sigma) staining by fluorescence-activated cell sorting (FACS) analysis. The cultured cells were harvested and washed in cold phosphate buffered saline (PBS), then fixed in 70% cold alcohol at 4°C overnight and washed with ice-cold PBS again. After stained with PI solution (40 mg/ml) at 37°C for 30 min in the dark, cells were analyzed on a Guava EasyCyte 5HT flow cytometer. The data were analyzed by Guava Incyte Software v2.2.2.
The apoptosis of U-2OS cells was measured by Guava Nexin Reagent (Millipore) according to the manufacture's protocol. The cultured cells were collected and resuspended in 100 μl medium supplemented with 1% FBS. The cells were then incubated with 100 μl Annexin V-PE and 7-AAD labeling solution (Millipore) for 20 min at room temperature in the dark. The resultant stained cells were analyzed on Guava EasyCyte 5HT flow cytometer. The data were analyzed by Guava Nexin Software v2.2.2.
The apoptosis of U-2OS cells was measured by Guava Nexin Reagent (Millipore) according to the manufacture's protocol. The cultured cells were collected and resuspended in 100 μl medium supplemented with 1% FBS. The cells were then incubated with 100 μl Annexin V-PE and 7-AAD labeling solution (Millipore) for 20 min at room temperature in the dark. The resultant stained cells were analyzed on Guava EasyCyte 5HT flow cytometer. The data were analyzed by Guava Nexin Software v2.2.2.
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