The number of apoptotic cells in each group of cSCC cells was determined using annexin V-FITC and propidium iodide (PI) staining kit (BD Biosciences), according to the manufacturer’s protocol. The staining patterns were analyzed using flow cytometry (BD Biosciences) and Cell Quest Software (BD Biosciences). Apoptotic cells in the tumor mass obtained from the xenograft mouse model were highlighted using a TACS2 TdT-DAB In Situ Apoptosis Detection Kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s protocol, and counted under a microscope.
Annexin 5 fitc and propidium iodide staining kit
Manufactured by BD
The Annexin V-FITC and propidium iodide (PI) staining kit is a laboratory tool used for the detection and quantification of apoptosis and cell viability. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the outer cell membrane during the early stages of apoptosis. The FITC-conjugated Annexin V binds to these exposed phosphatidylserine residues, allowing the identification of apoptotic cells. Propidium iodide (PI) is a fluorescent dye that is able to penetrate the cell membrane of dead or damaged cells, intercalating with the DNA and emitting a red fluorescence signal. The combined use of Annexin V-FITC and PI allows for the discrimination between viable, early apoptotic, and late apoptotic/necrotic cells.
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3 protocols using annexin 5 fitc and propidium iodide staining kit
1
Quantification of Apoptosis in cSCC
2
Apoptosis and Necrosis Analysis by Flow Cytometry
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After the treatment, the cells were harvested, washed, and fixed overnight in ethanol (70%) at −20 °C. The cells were pelleted, washed in chilled PBS, and resuspended in staining solution (50 μg/mL). Following treatment, cells were harvested and stained using an annexin V/FITC and propidium iodide (PI) staining kit (BD Biosciences), before incubating in the dark at room temperature (20–25 °C) for 15 min. Following staining, cells were analyzed by flow cytometry. A quadrant gate was applied (according to the manufacture’s protocol) to visualize all of the dot plots corresponding to the tests, and the percentages of cells in different quadrants were calculated.
3
Annexin V-FITC and PI Staining of Astrocytes
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The astrocytes were collected and stained using Annexin V-FITC and propidium iodide (PI) staining kit obtained from BD Biosciences as per the protocol provided by the manufacturer. Briefly, the astrocytes were resuspended and incubated with Annexin V-FITC and PI for 15 min. The dead cells were detected within 1 h using flow cytometry purchased from Becton Dickinson.
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