Fluorescein Isothiocyanate (FITC) Annexin V/PI Apoptosis detection kit II (BD PharmingenTM: 556570, the Scientific Group, Roodeport, South Africa) was used to detect apoptosis in treated MCF-7/DOX cells. After dark, PBM, PDT, and combination-treatment cells were harvested, washed twice, and stained with equal volume (5 µL) of FITC Annexin V and PI solution in 100 µL of a kit binding resolution. The mixture was incubated in the dark at room temperature for 15 min and analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, the Scientific Group, Roodeport, South Africa).
Annexin 5 fitc pi apoptosis detection kit 2
Manufactured by BD
Sourced in United States
The Annexin V-FITC/PI Apoptosis Detection Kit II is a laboratory tool designed to detect and analyze apoptosis, a programmed cell death process, in cell samples. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, and propidium iodide (PI), a DNA-binding dye, to differentiate between viable, early apoptotic, and late apoptotic/necrotic cells through flow cytometry or fluorescence microscopy.
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5 protocols using annexin 5 fitc pi apoptosis detection kit 2
1
Apoptosis Detection in Drug-Resistant Breast Cells
2
Apoptosis Detection in MCF-7/DOX Cells
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A fluorescein Isothiocyanate (FITC) Annexin V/PI Apoptosis detection kit II (BD PharmingenTM: 556570) was used to detect apoptosis in MCF-7/DOX cells. After PDT treatments, cells were harvested with trypLETM (Gibco 12563-029) and washed twice with cold PBS by centrifugation at 400× g in a 5 mL flow cytometer test tube before being suspended in 200 µL binding solution. After that, the cells were stained with a mixture of 5 µL of FITC Annexin V and 5 µL of PI. The tube was incubated in the dark at room temperature for 15 min. The mixture was analysed using a BD Accuri™ C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
3
Cell Cycle and Apoptosis Analysis
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To determine cell-cycle distribution, cells transfected with siNC or siSLC39A4 were fixed in 75% ethanol, washed twice with PBS, and incubated in 500 μL PBS containing 10 μL PI (1 mg/mL) and 0.5 μL RNase (10 mg/mL) at 37 °C for 30 minutes in the dark. Cell cycle was then assessed using flow cytometry (FACS Canto™ II, BD).
For apoptosis analysis, the siSLC39A4-transfected ESCC cells treated with CDDP for 24 hr, were collected and stained using Annexin V-FITC/PI Apoptosis detection Kit II (BD) for 15 minutes according to the recommendation of manufacturer. Then, the apoptosis rate was detected on FACS Canto™ II (BD).
For apoptosis analysis, the siSLC39A4-transfected ESCC cells treated with CDDP for 24 hr, were collected and stained using Annexin V-FITC/PI Apoptosis detection Kit II (BD) for 15 minutes according to the recommendation of manufacturer. Then, the apoptosis rate was detected on FACS Canto™ II (BD).
4
Quantifying Apoptosis, Cell Cycle, and Oxidative Stress
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A total of 2 × 105 cells was analyzed using an Annexin V-FITC/PI apoptosis detection kit II (BD Pharmingen™, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. To assess the distribution of nuclear DNA content, cells were collected, washed in PBS and fixed overnight in 75% ethanol at −20°C, treated with 1% RNase A for at least 15 min at 37°C, and stained with 50 μg/ml PI. For mitochondrial trans-membrane potential assessment, 1 × 106 cells were washed twice with PBS, incubated with 10 μg/mL of Rh123 for 30 min at 37°C, and stained with 50 μg/mL of PI. Mitochondrial cytochrome c was measured by FlowCellect™ cytochrome c kit (Millipore) according to manufacturer’s protocols. To measure ROS levels, 5 × 105 cells were washed with RPMI1640 and incubated with 5 μM DCFH-DA for 30 min at 37°C. The fluorescent intensity was measured by flow cytometry (Becton Dickinson, San Jose, CA, USA). All experiments were performed in triplicate and data were collected, stored, and analyzed by Lysis 11 software (Becton Dickinson).
5
Evaluating Cell Viability, Cycle, and Apoptosis
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Cell viability was measured by WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay according to the manufacturer's instruction (Roche). Briefly, after lentivirus infection, A375 and SK-MEL-28 cells were reseeded in a 96‑well plate, respectively, at a density of 1x104 cell/well. This assay entails the addition of 10 mL WST-1 reagents per 100 mL cell cultures in a 96-well plate. These cultures were incubated for 30 minutes and the absorbance at 450 nm was determined by an ELISA Microplate Reader (Bio-Rad).
In cell cycle assay, cells were collected and washed with pre‑cooling PBS and fixed with cooling 70% ethanol overnight at 4˚C. After washing twice with pre‑cooling PBS, the cells were resuspended and incubated in 200 μl PBS containing 50 μg/ml propidium iodide (PI) solution and 100 μg/ml RNase for 30 min at room temperature.
In apoptosis assay, cells were assessed with Annexin V‑FITC/PI apoptosis detection Kit II (BD) on a flow cytometer (BD Biosciences, San Diego, CA, USA) following the manufacturer's instruction. Data were analyzed using CellQuest software.
In cell cycle assay, cells were collected and washed with pre‑cooling PBS and fixed with cooling 70% ethanol overnight at 4˚C. After washing twice with pre‑cooling PBS, the cells were resuspended and incubated in 200 μl PBS containing 50 μg/ml propidium iodide (PI) solution and 100 μg/ml RNase for 30 min at room temperature.
In apoptosis assay, cells were assessed with Annexin V‑FITC/PI apoptosis detection Kit II (BD) on a flow cytometer (BD Biosciences, San Diego, CA, USA) following the manufacturer's instruction. Data were analyzed using CellQuest software.
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