The experiments were carried out according to our previous reports28 (link),36 (link). Cells were seeded into 6-well plate, after adherent cells were treated with siRNA for 48 h. Cells were collected by centrifugation. Cells used to detect cell cycle were resuspended with 500 μl PBS and added 2 ml 70% ethanol for fixation and permeabilization overnight. 50 μg/ml PI, 0.2% Triton-X-100, and 100 μg/ml RNase complex were applied in dark. Annexin V- FITC apoptosis analysis kit was purchased (SUNGENE BIOTECH) for test apoptotic cells. The sample was analyzed by flow cytometry.
Annexin 5 fitc apoptosis analysis kit
Manufactured by Sungene Biotech
Sourced in China
The Annexin V-FITC apoptosis analysis kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death, in cell samples. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule that becomes exposed on the cell surface during apoptosis. The Annexin V is conjugated with the fluorescent dye FITC, allowing for the visualization and measurement of apoptotic cells using flow cytometry or fluorescence microscopy.
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15 protocols using annexin 5 fitc apoptosis analysis kit
1
Cell Cycle and Apoptosis Analysis
2
Apoptosis Quantification by Flow Cytometry
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The rate of apoptosis was determined by flow cytometry in the dark after staining with an annexin V-FITC apoptosis analysis kit (Sungene Biotech Co., Tianjin, China).
3
Quantifying Apoptosis with Annexin V-FITC
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The Annexin V-FITC Apoptosis Analysis kit (Tianjin Sungene Biotech, China) was used to assess cell apoptosis. Cells were collected using EDTA-free pancreatic enzyme, washed with cold PBS, and resuspended in 1× binding buffer. Annexin V-FITC and PI solutions were added to the cells and incubated in the dark for 20 min at room temperature.
4
Apoptosis Analysis of SH-SY5Y Cells
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After treatment with drugs, differentiated SH-SY5Y cells were collected and resuspended in phosphate buffer solution. An Annexin V-FITC apoptosis analysis kit (Sungene Biotech, Tianjin, China) was used to identify apoptotic cells. Next, the apoptotic rate was determined using CytoFLEX (Beckman Coulter, California, USA).
5
Annexin V-FITC Apoptosis Assay
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Cell apoptosis was assessed with an Annexin V-FITC Apoptosis Analysis Kit (AO2001-02A-H, Sungene Biotech, China). After collecting, the cells were washed twice with cold PBS and re-suspended in 100 μl of 1 × Annexin binding buffer. Five microliters Annexin V-FITC and 5 μl 7-AAD solutions were then added to the cell suspension and incubated at 37 °C for 15 min. The stained cells were analysed with a FACS system (FACSAria, BD Bioscience).
6
Apoptosis Assay in C2C12 Myoblasts
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Apoptosis was assessed using an Annexin V-FITC Apoptosis Analysis Kit (Tianjin Sungene Biotech Co., Ltd.). C2C12 myoblasts were seeded in a 12-well plate at a density of 2 × 105 cells per well and treated with or without 30 μg/mL APN for 24 h. Then, the cells were treated in the presence or absence of 5 mM H2O2 for 30 min. The cell pellets were resuspended in 100 μL buffer with 5 μL annexin V-FITC for 10 min and then incubated with 5 μL propidium iodide for 5 min at room temperature in the dark. A total of 500 μL buffer was added, and the cells were immediately analyzed using an Accuri C6 (BD, CA, USA). A total of 10,000 cells per sample were acquired, and percentage of cell death was analyzed.
7
Annexin V-FITC Apoptosis Assay
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Cell apoptosis was detected with flow cytometry (Beckman Coulter, Brea, CA, USA) according to the instructions provided in Annexin V-FITC apoptosis analysis kit (Tianjin Sungene Biotech Co., Ltd., Tianjin, China). The details of the materials and methods are shown in the Section S1 .
8
Annexin V-FITC Apoptosis Assay
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Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC apoptosis analysis kit (AO-2001-02P-H, Tianjin Sungene Biotech Co., Ltd, Tianjin, China). After the four group of cells were cultured under the condition as described above for 24 hours, they were digested by trypsin, collected, and washed three times with PBS diluted 1×binding buffer (1ml per tube). For each sample with 100 μL cell suspension, 5 μL (2.5 μg/mL) of Annexin-V-FITC were added, and after gentle vortex the samples were incubated for 10 min at room temperature in the dark. Then 5 μL of PI solution were added and incubated at the same condition. After being supplemented to 500 μL using cold PBS followed by gentle vortex, samples were ready to be testes. Apoptosis signals were detected using a CytoFLEX by Beckman & Coulter.
9
Annexin V-FITC Apoptosis Assay
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Apoptotic and necrotic cells were detected by the Annexin V-FITC apoptosis analysis kit (Tianjin Sungene Biotech, China). Different genotypes of cells density were adjusted to 5 × 106 cells/ml. About 100 μl cell suspension was incubated with 2.5 μl AnnexinV/FITC for 10 min and then 2.5 μl PI for 5 min at room temperature in dark. The rate of apoptosis was measured by flow cytometry (BD Pharmingen).
10
Quantifying Apoptosis by Annexin V-FITC and PI
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The percentages of viable (annexin-/propidium iodide-PI-) and early apoptotic (annexin+/PI-) cells were quanti ed by using annexin V-uorescein isothiocyanate (FITC) staining, which was used to detect phosphatidylserine that is externalized in the early phases of apoptosis. Annexin V is an important marker of early apoptosis in which changes in externalized phosphatidylserine levels occur prior to DNA fragmentation [14] .We used the Annexin V-FITC Apoptosis Analysis Kit according to the manufacturer's instructions (Tianjin Sungene Biotech Co., Ltd., China); 1×10 5 mononuclear cells were labeled, incubated in the dark for 15 min and immediately sorted by owcytometry (BD FACSAria TM ш, USA). Marked annexin V-FITC staining (green) was analyzed using the FL-1channel, while PI staining (red) was analyzed using the FL-2 channel. The data were analyzed by using the owjo software on a BD FACS Aria™ cytometer (BD BioSciences, USA).
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