For FITC-annexin V staining, 1.0×106 cells were suspended in 100 µl annexin staining buffer and 5 µl FITC-Annexin V solution (BD Biosciences) was added to the suspension. After 15 min, the volume of suspension was made up to 500 µl with annexin staining buffer. For PI staining, 5 µl of 100 µg/ml PI solution was added to DC suspensions. Cells were immediately analyzed by flow cytometry.
Annexin 5 fitc solution
Manufactured by BD
Sourced in United States
Annexin V-FITC solution is a laboratory reagent used in cell biology research. It contains Annexin V, a calcium-dependent phospholipid-binding protein, conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate). This solution is used to detect and quantify apoptosis (programmed cell death) in cell samples.
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Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Pi staining solution, by BD (1 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Modfit lt 3, by Verity Software House (1 mentions)
Annexin binding buffer, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Facsverse, by BD (1 mentions)
9 protocols using annexin 5 fitc solution
1
FITC-Annexin V Apoptosis Assay
2
Apoptosis Detection by Annexin V-FITC/PI
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Cells were collected by centrifugation and suspended with Annexin V binding buffer (BD Biosciences). Then 5 μl of FITC‐Annexin V solution (BD Biosciences) was added and incubated for 15 min, and stained using 5 μl PI staining solution (BD Biosciences) at room temperature for 5 min. Finally, the fluorescence signals of the PI channel and FITC channel were analyzed by flow cytometry.
3
Quantifying Apoptosis using Annexin V-FITC and PI
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To quantify apoptosis cells were harvested and washed in ice-cold PBS. After centrifugation at 4°C for 5 min, cells were resuspended in annexin V-binding buffer (10 mM HEPES; pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells were labeled with annexin V-FITC solution (BD Biosciences, San Jose, CA) and/or propidium iodide (PI; 10 μg/ml) for 15 min at room temperature in the dark. PI is impermeable to living and apoptotic cells, but stains necrotic and apoptotic dying cells with impaired membrane integrity, in contrast to annexin V, which stains early apoptotic cells. Quantification of positive cells was done in a FACSCanto II flowcytometer (BD). Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-O-methyl-fluoromethylketone) was used at 20 μM to inhibit caspases.
4
Cell Cycle and Apoptosis Analysis of Glioma Cells
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Cell-cycle detection: the glioma cells were fixed overnight in 70% ethanol solution (v/w) at 4 °C, and stained in propidium iodide (PI) the next day at 37 °C for 30 minutes. The cell cycle was detected on a FACScalibur flow cytometer (BD, Franklin Lakes, NJ, USA), and estimated using Mod Fit LT3.1 software (Verity Software House, Topsham, ME, USA).
Apoptosis detection: after centrifugation, the cells were collected and counted, and 5×105 cells were centrifuged at 1,000 rpm at 4 °C for 10 minutes, and the supernatant was removed. Afterward, 1 mL of precooled PBS was added to the cells, and the tube slightly shaken. After centrifugation for 2 times at 1,000 rpm at 4 °C, the supernatant was discarded and cells were resuspended in 200 µL binding buffer. Next, the cells were mixed with 10 µL of annexin-V-FITC solution (51-65874X, BD) for 15 minutes without light exposure. Finally, the cells were treated with 300 µL of combined buffer and 5 µL of PI, and tested on the flow equipment.
Apoptosis detection: after centrifugation, the cells were collected and counted, and 5×105 cells were centrifuged at 1,000 rpm at 4 °C for 10 minutes, and the supernatant was removed. Afterward, 1 mL of precooled PBS was added to the cells, and the tube slightly shaken. After centrifugation for 2 times at 1,000 rpm at 4 °C, the supernatant was discarded and cells were resuspended in 200 µL binding buffer. Next, the cells were mixed with 10 µL of annexin-V-FITC solution (51-65874X, BD) for 15 minutes without light exposure. Finally, the cells were treated with 300 µL of combined buffer and 5 µL of PI, and tested on the flow equipment.
5
Apoptosis Evaluation Protocol for T. cruzi Extract
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The apoptosis evaluation was performed as described in [40 (link)]. The splenic cells (1 × 106/ml) in 96-well plates (100 µl/well) were incubated for 24 h, 37 °C, 5% CO2 with T. cruzi extract (300 µg/ml) or RPMI (control). In parallel, the same approach was performed for 15 min. After incubation, the cells were transferred to 12 × 75 mm round-bottomed polystyrene tubes (100 µl/tube), washed twice with PBS and centrifuged for 3 min, 2500×g, 4 °C. The cells were suspended in Annexin-V FITC solution (1 µl Annexin-V FITC in 100 µl Annexin Binding Buffer, BD Biosciences Pharmingen) and incubated for 15 min, at room temperature, in the dark. Before the flow cytometry analysis, the cells received propidium iodide (PI, BD Biosciences Pharmingen). Early (Annexin+PI−) and late (Annexin+PI+) apoptosis were determined after analysis in a FACSCanto flow cytometer (BD Biosciences) equipped with the FACSDiva software.
6
Apoptosis Assay for HUVECs and H9C2
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HUVECs or H9C2 myocytes were washed in PBS and resuspended at the density of 5 × 105 cells/mL with 500 μL binding buffer (1×). Afterwards, 5 μL Annexin V‐FITC solution and 5 μL propidium iodide (BD, NY, USA) were added into the suspension and the mixture was incubated for 15 minutes at room temperature avoiding light. Then, cells were performed with flow cytometry analysis.
7
Apoptosis Assessment by Flow Cytometry
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After the various treatments were completed, the cells (1×106) in each group were assessed for apoptosis using flow cytometry. Briefly, the detection process was as follows: Each group of cells was co-incubated with propidium iodide (PI) and Annexin V-FITC solution (BD Biosciences) and processed according to the instructions of the kit. The apoptosis level of each group of cells was examined by flow cytometry (BD FACSVerse™; BD Biosciences) and the results were analyzed by FlowJo (Flow Jo 7.6; Tree Star Inc.).
8
Annexin V-FITC Apoptosis Assay
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The cells transfected for 48 hrs were inoculated into 6-well plates (1×105 cells/well). After being washed with PBS, a total of 200 μL cells were incubated in dark with 2 μl Annexin V-FITC solution (BD Bio., USA) and 1 μL of PI solution (30 mins). Then, the apoptosis was recorded by flow cytometry.
9
Annexin-V Apoptosis Assay in SSC-4 Cells
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SSC-4 cells were seeded at a density of 0.5×106 cells in six-well plates in 2 mL culture media and treated for 24 and 48 hours with a single dose of 20 μM. The cells were harvested, then washed once with phosphate-buffered saline (PBS) and centrifuged at 500 RPM for 5 minutes. Then, the SSC-4 cells were resuspended and stained with Annexin- V-FITC solution (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 15 minutes at room temperature. Just before analysis, it was stained with propidium iodide (PI) in order to quantify the necrotic cells. Apoptotic and necrotic cells were evaluated on a BD FACSCanto™ II flow cytometer.
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