After 48 h of culture, 0.25% trypsin without EDTA was added to digest the transfected CNE‐2 cells. The cells were washed twice with phosphate buffered saline, centrifuged at 1500 g for 3 min, and the supernatant was removed. An Annexin V‐FITC/PI apoptosis detection kit (Tianjin Sungene Biotech Co., Ltd, Tianjin, China) was used as follows: 500 μL binding buffer was added to resuspend the cells, followed by 5 μL Annexin V‐FITC and 5 μL PI. The tube was mixed well and then incubated and protected from light at room temperature for 5–15 min. Cell apoptosis was detected using flow cytometry and statistically analyzed by an independent‐samples t‐test (the upper right quadrant should be the late apoptotic cell, and the lower right quadrant should be the early apoptotic cell). All the experiments were repeated three times.
Annexin 5 fitc pi apoptosis detection kit
Manufactured by Sungene Biotech
Sourced in China
The Annexin V-FITC/PI apoptosis detection kit is a lab equipment product that allows for the detection and analysis of apoptosis in cell samples. It utilizes Annexin V, a protein that binds to phosphatidylserine, and propidium iodide, a DNA-binding dye, to differentiate between viable, early apoptotic, and late apoptotic/necrotic cells.
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7 protocols using annexin 5 fitc pi apoptosis detection kit
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Annexin V-FITC/PI Apoptosis Assay
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Apoptosis Detection in AML Cells
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Apoptosis of KG-1a cells and primary human AML samples was detected with an Annexin V-FITC/PI Apoptosis Detection Kit (SUNGENE BIOTECH, China). KG-1a cells were cultured in 24-well plates at a density of 1 × 105cells/well for 24 h and treated with CPPTL alone or together with 5 mM NAC. Primary human AML samples were cultured in 24-well plates treated with CPPTL for 24 h, at a density range from 2×105 to 5×105cells/well depending on the total cell numbers of different samples. After 24 h, cells were washed with cold phosphate-buffered saline (PBS), re-suspended in 1×binding buffer and incubated with Annexin V-FITC in the dark for 10 min. PI was added 5 min before detection. All samples were analyzed with a BD LSRFortessa flow cytometer (BD Biosciences, USA).
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Apoptosis Detection in Hepatoma Cells
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An Annexin V-FITC/PI apoptosis detection kit (Sungene Biotech, cat. No. AO2001-02P-H) was used to detect cell apoptosis by flow cytometry as described previously25 (link). Hepatoma cells were seeded in 6-well plates. Then the cells were cultured for 24 h in complete medium. Afterward, the cells were incubated with sorafenib and bilirubin for 24 h. The medium was discarded, and the cells were washed with PBS twice. After trypsinization for 1 min, cells were collected and resuspended in binding buffer, and PI and Annexin V-FITC were added according to the manufacturer’s protocol, and incubated for 15 min in the dark. Finally, apoptotic cells were detected with a flow cytometer (Becton Dickinson Biosciences).
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Annexin V-FITC/PI Apoptosis Assay
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Annexin V-FITC/PI apoptosis detection kit (Sungene Biotech, Tianjin, China) was used to detect the effect of SSd on AR42J apoptosis according to the manufacturer’s instruction. The samples were measured by a FACScan flow cytometer (Beckman, CA, United States) as previously described (Cui, et al., 2020).
5
Cell Cycle, Apoptosis, and ROS Analysis
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For analysing the cell cycle, cells were trypsinized and fixed in 70% cold ethanol at −20 °C. Before performing flow cytometry, cells were washed 3 times in PBS and treated with a final concentration of 0.2 mg/mL aRNase and 50 μg/mL propidium iodide (PI) for 30 min at 37 °C and protected from light. For the analysis of apoptosis, cells were trypsinized without EDTA. Apoptotic cells were monitored using the Annexin V-FITC/PI apoptosis detection kit (Tianjin Sungene Biotech Co). For ROS detection, cells were incubated with 10 μM 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) for 30 min at 37 °C, then trypsinized and resuspended in PBS. Flow cytometry was performed on the Guava easyCyte HT system (Millipore Corporation).
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Annexin V-FITC/PI Apoptosis Assay
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Apoptosis was measured with an Annexin V-FITC/PI Apoptosis Detection Kit (Tianjin Sungene Biotech, China). Cells were collected and suspended with 400 μl 1 × binding buffer. Then, 5 μl of Annexin V-FITC was added to the cell suspension and incubated at 2–8°C for 15 min. After that, 10 μl of PI was added and incubated for 5 min at 2–8°C in the dark. Finally, cells were analyzed by flow cytometry (BD FACS Vantage SE). Annexin V-positive/PI-positive cells were considered as late apoptotic or necrotic cells.
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Annexin V-FITC/PI Apoptosis Assay for BRL Cells
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To detect the apoptosis of BRL cells treated with SeNPs, an Annexin V-FITC/PI apoptosis detection kit (Tianjin Sungene Biotech Co., Ltd., Tianjin, China) was used according to the manufacturer's instruction. The BRL cells were seeded in 6-well plates. After incubation with different concentrations of SeNPs, cells were collected into 10 mL centrifugal tubes and washed twice with cold PBS, then centrifuged for 5 min at 1000 rpm at 4°C. Cells were suspended with 1 × binding buffer at a concentration of 1 × 106 cells/mL in 10 mL centrifugal tubes. Next, 100 μL of resuspended cells was added into new 10 mL centrifugal tubes. Then, 5 μL Annexin V-FITC was added into the tubes and incubated at room temperature in the dark for 10 min. Then, 5 μL of propidium iodide (PI) was added into the tubes and incubated at room temperature in the dark for 5 min. Finally, PBS was added to 500 μL. The samples were immediately analyzed by a flow cytometer (BD Biosciences, San Jose, CA, USA) with an excitation wavelength of 488 nm in 1 h.
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