Cells were labeled by FITC-coupled Annexin V (Annexin V-FITC) (BD Biosciences, NJ, USA) for detection of phosphatidylserine exposure and by propidium iodide (PI) for observation of the loss of membrane integrity. Cells were harvested by trypsinization and washed twice with ice-cold phosphate buffer saline (PBS). Then, cells were resuspended in 1× binding buffer at a concentration of 1 × 106 cells/mL. Transferred 100 μL of the solution (1 × 105 cells) to a 5 mL culture tube and stained with 5 μL Annexin V-FITC and 5 μL propidium iodide (PI). The suspension was incubated at room temperature in the dark for 15 min. Added 400 μL of 1× binding buffer to each tube. FACS Calibur Flow Cytometer (BD Biosciences, NJ, USA) was used to distinguish cells and the results were analyzed by FlowJo software (Tree Star Corp, Ashland, OR). All experiments were independently repeated three times.
Annexin 5 annexin 5 fitc
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Annexin V is a protein that binds to phosphatidylserine, a phospholipid found on the surface of cells undergoing apoptosis (programmed cell death). Annexin V-FITC is a fluorescently labeled version of Annexin V, which allows for the detection and quantification of apoptotic cells using flow cytometry or fluorescence microscopy.
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5 protocols using annexin 5 annexin 5 fitc
1
Annexin V-FITC and PI Apoptosis Assay
2
Annexin V-FITC/PI Apoptosis Assay
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Cells were labeled by FITC-coupled Annexin V (Annexin V-FITC) (BD Biosciences, NJ, USA) for detection of phosphatidylserine exposure and by propidium iodide (PI) for observation of the loss of membrane integrity. Cells were harvested by trypsinization and washed twice with ice-cold phosphate buffer saline (PBS). Then, cells were resuspended in 1×binding buffer at a concentration of 1×10 6 cells/ml. Transferred 100μl of the solution (1×10 5 cells) to a 5ml culture tube and stained with 5μl Annexin V-FITC and 5μl propidium iodide (PI). The suspension was incubated at room temperature in the dark for 15min. Added 400μl of 1×binding buffer to each tube. FACS Calibur Flow Cytometer (BD Biosciences, NJ, USA) was used to distinguish cells and the results were analyzed by FlowJo software (Tree Star Corp, Ashland, OR). All experiments were independently repeated three times.
Transmission electron microscopic examination PC12 cells were xed in 2.5% glutaraldehyde and rinsed with 0.1mol/L phosphate-buffered saline (PBS). Samples were placed in 1% osmium acid at 4°C for 4h. After that, they were dehydrated in ethanol, embedded in the embedding agent, and stained with uranyl acetate and lead citrate. Finally, samples were examined under a transmission electron microscope (JEM-1400, JEOL, Japan).
Transmission electron microscopic examination PC12 cells were xed in 2.5% glutaraldehyde and rinsed with 0.1mol/L phosphate-buffered saline (PBS). Samples were placed in 1% osmium acid at 4°C for 4h. After that, they were dehydrated in ethanol, embedded in the embedding agent, and stained with uranyl acetate and lead citrate. Finally, samples were examined under a transmission electron microscope (JEM-1400, JEOL, Japan).
3
Annexin V-FITC/PI Apoptosis Assay
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Cells were labeled by FITC-coupled Annexin V (Annexin V-FITC) (BD Biosciences, NJ, USA) for detection of phosphatidylserine exposure and by propidium iodide (PI) for observation of the loss of membrane integrity. Cells were harvested by trypsinization and washed twice with ice-cold phosphate buffer saline (PBS). Then, cells were resuspended in 1×binding buffer at a concentration of 1×10 6 cells/ml. Transferred 100μl of the solution (1×10 5 cells) to a 5ml culture tube and stained with 5μl Annexin V-FITC and 5μl propidium iodide (PI). The suspension was incubated at room temperature in the dark for 15min. Added 400μl of 1×binding buffer to each tube. FACS Calibur Flow Cytometer (BD Biosciences, NJ, USA) was used to distinguish cells and the results were analyzed by FlowJo software (Tree Star Corp, Ashland, OR). All experiments were independently repeated three times.
Transmission electron microscopic examination PC12 cells were xed in 2.5% glutaraldehyde and rinsed with 0.1mol/L phosphate-buffered saline (PBS). Samples were placed in 1% osmium acid at 4°C for 4h. After that, they were dehydrated in ethanol, embedded in the embedding agent, and stained with uranyl acetate and lead citrate. Finally, samples were examined under a transmission electron microscope (JEM-1400, JEOL, Japan).
Transmission electron microscopic examination PC12 cells were xed in 2.5% glutaraldehyde and rinsed with 0.1mol/L phosphate-buffered saline (PBS). Samples were placed in 1% osmium acid at 4°C for 4h. After that, they were dehydrated in ethanol, embedded in the embedding agent, and stained with uranyl acetate and lead citrate. Finally, samples were examined under a transmission electron microscope (JEM-1400, JEOL, Japan).
4
Apoptosis Analysis of Coloaded Nanoparticles
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L02 and SK-Hep-1 (1×106 cells/well) were seeded in six-well plates and incubated at 37°C for 24 hours. After incubation with blank NPs (HP and SHP) or coloaded NPs (FasudilHPmiR195, FasudilSHPmiR195) for another 24 hours, apoptosis analysis was performed using the Annexin V-FITC (Annexin V) and propidium iodide detection kit (BD Pharmingen), according to the manufacturer’s instructions. The untreated cells were used as control.
5
Apoptosis Analysis of PC-12 Cells
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Flow cytometry, Annexin V-FITC (Annexin V) and PI (propidium iodide) (BD Biosciences, NJ, USA) were used to analyse the apoptosis of the PC-12 cells. After induction by OGD and Remifentanil in vitro, and transfection for 48 h, the PC-12 cells (1 × 10 6 cells per well) in a 6-well cell-culture plate were washed twice with PBS, and then stained for 15 min in darkness, at room temperature, with Annexin V-FITC. Finally, apoptosis was analysed by ow cytometry, including early apoptosis (Annexin V positive / PI negative in the fourth quadrant).
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