Cells were plated in 6-well plates, and 48 h after transfection, the cells were collected and resuspended in staining solution containing Annexin V-FITC and propidium iodide (PI; Tianjin Sungene Biotech Co. Ltd., Tianjin, China). The stained cells (1 × 10 5 ) were analyzed using a flow cytometer.
Annexin 5 fitc
Manufactured by Sungene Biotech
Sourced in China
Annexin V-FITC is a fluorescent conjugate of the protein annexin V. Annexin V has a high affinity for the phospholipid phosphatidylserine, which is exposed on the surface of apoptotic cells. When Annexin V is conjugated with the fluorescent dye FITC, it can be used to detect and quantify apoptotic cells in flow cytometry and other cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Sungene Biotech (3 mentions)
Facscanto 2, by BD (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
α lipoic acid, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Flow cytometry, by BD (1 mentions)
Fulvestrant, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Gefitinib, by AstraZeneca (1 mentions)
Facsdiva software, by BD (1 mentions)
Annexin 5 apc, by Sungene Biotech (1 mentions)
Flow cytometry analysis, by Beckman Coulter (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Aria 3, by BD (1 mentions)
Annexin 5 apoptosis detection kit, by Sungene Biotech (1 mentions)
Anti cd8 pe cy7, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by BioLegend (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
14 protocols using annexin 5 fitc
1
Apoptosis Quantification by Flow Cytometry
2
Oxidative Stress Evaluation Protocol
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The Zymuphen MP-Activity ELISA kit was purchased from HYPHEN BIOMED, U.S.A. Antioxidants N-Acetyl-l -cysteine (NAC) and α lipoic acid (α-LA) were purchased from Sigma, U.S.A. The probe of CM-H2DCFDA and MitoSOX™ Red were purchased from Molecular Probes, U.S.A. Annexin V-FITC was purchased from Tianjin Sungene Biotech Co. Ltd. Antibodies against NOX4 was purchased from Cell Signaling Technology, U.S.A. PCR primers were synthesized by Takara.
3
Cell Cycle and Apoptosis Analysis in HeLa and SiHa Cells
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Cell cycle and apoptosis analysis were carried out to detect the effect of LONP2 on HeLa and SiHa cells. For cell cycle studies, cells were harvested48 h after transfection, resuspended with 100 μl PBS, and centrifuged. Then, cells were fixed in cold ethanol and incubated overnight at 4°C. After washing twice with PBS, we incubated the cells with 500 μl of RNase/PI (Tianjin Sungene Biotech Co., Ltd.) for 20 min at room temperature away from light, and then used FACSCalibur (BD) for analysis.
For the apoptosis assay, at 48 h after transfection, cells were harvested, centrifuged, and washed with PBS. Then, cells were resuspended with 300 μl binding buffer, stained with 5 μl Annexin V-FITC (Tianjin Sungene Biotech Co., Ltd), and incubated for 15 min in the dark. We then added 5 μl PI, and the cells were incubated for 5 min in the dark and assessed with FACSCalibur (BD).
For the apoptosis assay, at 48 h after transfection, cells were harvested, centrifuged, and washed with PBS. Then, cells were resuspended with 300 μl binding buffer, stained with 5 μl Annexin V-FITC (Tianjin Sungene Biotech Co., Ltd), and incubated for 15 min in the dark. We then added 5 μl PI, and the cells were incubated for 5 min in the dark and assessed with FACSCalibur (BD).
4
Annexin V-FITC and PI Apoptosis Assay
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Cells were seeded into 6-well plates and treated with Gefitinib (AstraZeneca) after transfection or combined with Fulvestrant (ICI 182,780, Sigma-Aldrich) for 48 h. Cells in each well were harvested, washed and resuspended in 1× binding buffer, and then stained with Annexin V-FITC and PI (Sungene Biotech, China) for 10 min in the dark, respectively. Data acquisition was performed on a flow cytometry (Becton–Dickinson, USA) with the CellQuest software (BD Biosciences, USA).
5
Annexin V-Based Apoptosis Detection
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Annexin V-FITC (AO2001-10) and Annexin V-APC (AO2001-11) Apoptosis Analysis Kits (Sungene Biotech, Tianjin, China) were used to detect cell apoptosis. Cell death detection was performed following the manufacturer’s instruction. Briefly, cells were harvested, washed with cold PBS, suspended in 1× binding buffer, and then centrifuged at 300 × g for 10 min. Supernatants were removed, and cells were resuspended in 100 μL of binding buffer with addition of 5 μL of Annexin V-FITC (APC), and after a gentle vortex, they were incubated for 10 min in room temperature, and then another 5 min in room temperature after addition of 5 μL of PI solution. Flow cytometry was performed by using a FACSSanto II (BD Biosciences), and data were analyzed with FACSDiva software (BD Biosciences).
6
Cell Cycle, Apoptosis, and ROS Analysis
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Cells were collected and washed three times with PBS. For the cell cycle analysis, cells were re-suspended in 1 mL DNA staining solution. 10 μL propidium iodide (PI) solution (MultiSciences Biotech, China) was added. The cells were incubated in darkness for at least 30 min. For the apoptosis analysis, cells were re-suspended in 100 μL 1× Annexin V binding buffer, and then, we sequentially added 5 μL Annexin V-FITC and 5 μL PI solution (Sungene Biotech, China). Another 400 μL 1× Annexin V binding buffer was added after 30 min of incubation. A parameters regulation was performed at each independent experiment according to the user’s instructions. For the ROS analysis, cells were re-suspended in 1 mL FBS-free medium, and 1 μL DCFH-DA (Sigma, D6883) was added. After 30 min of incubation, cells were centrifuged and re-suspended in a 1 mL FBS-free medium. Each sample was analyzed using flow cytometry analysis (Beckman, USA).
7
Apoptosis Evaluation in SK-N-SH Cells
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Before the cultivation of cell suspension (1 × 10 5 cells) with 5 μL Annexin V-FITC (Tianjin Sungene Biotech Co., Tianjin, China) for 15 min and 5 μL PI (Tianjin Sungene Biotech Co., Tianjin, China) for 5 min at room temperature, treated SK-N-SH cells were harvested by trypsinization, washed with precold PBS, and resuspended in binding buffer. Cellular fluorescence was observed under flow cytometry (Sysmex Partec GmbH, Goerlitz, Germany).
8
Annexin V-FITC and PI Apoptosis Assay
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Precooled PBS was added at 4 °C, and an appropriate amount of binding buffer was diluted for use. After washing with PBS once, the cells in the 6-well plate were digested with 400 μL of 0.25% trypsin. The digestion was terminated by adding complete medium once the cells had become round and some had fallen off. We collected samples in 1.5 mL EP tubes and centrifuged them at 300 × g for 5 min, discarding the supernatant. One milliliter of PBS was added to resuspend the cells, they were centrifuged at 300 × g for 5 min, and the supernatant was discarded. A 200 μL solution of Binding Buffer was used to resuspend the pellet. Following this, 5 μL of Annexin V-FITC (Sungene Biotech, Tianjin, China) were added, mixed well and incubated for 10 min in the dark. Next, 5 μL propidium iodide was added, mixed well and incubated in the dark for 5 min. On-board inspection was performed within 1 h.
9
Cell Proliferation and Apoptosis Analysis
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The proliferation of NCI-H1299 and A549 cells after transfection was analyzed by fluorescence-activated cell-sorting (FACS) assay. For each assay, 100 µL of cells at a density of 1×105 were incubated with Annexin V-FITC and propidium iodide (Tianjin Sungene Biotech Co. Ltd., Tianjin, China) in the dark at 4 °C for 10 min. The cells were then washed twice with buffer and suspended in 500 µL of buffer for analysis by flow cytometry.
10
Annexin V-FITC Cell Viability Assay
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After the cells were centrifuged for 5 min at room temperature and resuspended in PBS at 4°C and washed by centrifugation, they were suspended in binding buffer diluted with deionized water and incubated for 15 min at room temperature in the dark by adding annexin V-FITC (AO2001-02P-G, Sungene Biotech) to the PI label, and the diluted binding buffer was added and then used for detection on the machine (AriaIII, BD).
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