Apoptosis was evaluated by Annexin V assay (Annexin V-FITC Kit, TREVIGEN INC.) following manufacturer protocol. Briefly, 5 × 105 cells were washed with cold PBS and incubated in 100 μL Annexin V Incubation Reagent for 15 min at room temperature in the dark. After staining, cells were analyzed by using a BD FACSCanto II (BD BIOSCIENCES; San Jose, CA USA). At least 10,000 events were counted for each sample to ensure statistical relevance.
Annexin 5 fitc kit
Manufactured by Bio-Techne
Sourced in United States
The Annexin V-FITC Kit is a laboratory tool used to detect and quantify apoptosis, a process of programmed cell death. The kit contains Annexin V, a protein that binds to phosphatidylserine, a molecule exposed on the surface of cells undergoing apoptosis. 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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Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Facsdiva software, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Facscan flow cytometer, by Tree Star (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
9 protocols using annexin 5 fitc kit
1
Annexin V Apoptosis Assay
2
Apoptosis Analysis of JAK2 and CALR Mutations
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Apoptosis was evaluated by Annexin V assay (Annexin V-FITC Kit, Trevigen Inc, Minneapolis, MN, USA) following manufacturer protocol. Briefly, 5 × 105 CD34+ cells from 5 JAK2 and 5 CALR-mutated samples were washed with cold PBS and incubated in 100 μL Annexin V incubation reagent for 15 min at room temperature in the dark. After staining, cells were analyzed by using a BD FACSCanto II (BD Biosciences; San Jose, CA, USA). At least 10,000 events were counted for each sample to ensure statistical relevance.
3
Identifying Cell Death Mechanism of DLBCL by ZnO NPs
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To determine the type of killing of the DLBCL cells induced by the ZnO NPs, the DLBCL cells in RPMI 1640 medium with 10% FBS (3×105 cells/mL) were incubate in a 6-well plate with and without ZnO NPs (30 μg/mL) for 24 hours at 37 °C in CO2 incubator. The type of cell death was investigated using FACS CantoII flow cytometer (Becton Dickinson) with an Annexin V-FITC Kit (Trevigen, Inc.). Briefly, a washing step of the malignant cells (3×105 cells/mL) with cold 1X phosphate buffered saline (1X PBS) followed by centrifugation for 5 minutes at 300×g was done. Next, a dye solution (fluorescein-labeled Annexin V, propidium iodide and 1X binding buffer) was used to stain the cells for 15 minutes at room temperature in dark. Next, the stained cells were transferred into a FACS tube and 1X binding buffer was added to them. Finally, the cells were injected into FACS CantoII flow cytometer (Becton Dickinson) and analyzed using FACSdiva software (Becton Dickinson). The experiments were performed three times independently.
4
Analyzing ZnO NPs-Induced Cell Death in K562 Cells
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To identify the kind of cell death induced by ZnO NPs in K562 cells, suspensions of the leukemic cells (3×105 cells/mL) in RPMI 1640 supplemented with 10% FCS were seeded into a 6-well plate in the absence of ZnO NPs (control cells) and in the presence of 40 μg/mL of ZnO NPs. The plate was then incubated for 3 days at 37 °C with the presence of CO2. The incubation of the cells was contiguous with no change of the culture medium. FACS analysis of cellular death was carried out using an Annexin V-FITC Kit (Trevigen, Inc.) at two time points of the incubation time (15 hours and 72 hours). In brief, cells (3×105 cells/mL) were washed with cold 1X phosphate buffered saline (1X PBS) and centrifuged for 5 minutes at 300×g. Next, washed cells were resuspended in a staining solution containing fluorescein-labeled Annexin V, propidium iodide and 1X binding buffer. The cells in the staining solution were incubated for 15 minutes in the dark at room temperature. Next, 1X binding buffer was added to the stained cells that were transferred into a FACS tube for subsequent analyses. The stained cells were then analyzed using FACS CantoII flow cytometer coupled with FACSdiva software (Becton Dickinson). The experiments were performed in triplicate.
5
Cell Viability Assay using Annexin V-FITC and PI
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The cell viability was determined by flow cytometry using the Annexin V-FITC kit (Trevigen, Gaithersburg, MD, USA). MCF-7 and T-84 cells were seeded at a high density (2 × 105 cells/cm2) in 6-well plates. After 24 h, the cells were induced with PTS and PTSO for 48 h at the IC50 concentration for each cell line. The cells were detached with the TrypLE Express Enzyme (ThermoFisher Scientific, Waltham, MA, USA), washed with PBS, and collected by centrifugation at 300× g for 10 min. Then, the cells were washed again and incubated with annexin-V FITC and propidium iodide (PI) in an annexin-V binding buffer for 15 min. After incubation, the cells were diluted with the binding buffer and examined immediately in a FACScan flow cytometer, using FlowJo (v.7.6.5, Tree Star, Inc., Ashland, OR, USA). This assay was performed in duplicate.
6
Quantifying Cellular Apoptosis and Necrosis
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The number of FSK cells undergoing apoptosis and/or necrosis after chronic exposure to 250 μM and 500 μM FA was quantified using an Annexin V-FITC kit (Trevigen, Gaithersburg, MD, USA), according to the manufacturer’s instructions. Briefly, 1 × 104 cells under different experimental conditions were collected by trypsinization, washed in PBS, resuspended in binding buffer, and mixed with Annexin V-FITC and propidium iodide (PI). After a 15 min 25 oC incubation in the dark, the cells were analyzed using a flow cytometer LSR II flow cytometer (BD Biosciences LSRFortessa, San Jose, CA, USA) and analyzed using Facsdiva software (BD Biosciences). Each experiment was performed in duplicate, repeated at least three times using different cell batches.
7
Apoptosis Evaluation by Annexin V Assay
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Apoptosis was evaluated by Annexin V assay (Annexin V-FITC Kit, Trevigen Inc.) as previously described [36 (link)]. Briefly, 5 × 105 cells were washed with cold PBS and incubated in 100 μL Annexin V Incubation Reagent for 15 min at room temperature in the dark. After staining, cells were analyzed by using a BD FACSCanto II (BD Biosciences; San Jose, CA USA). At least 100,000 events were counted for each sample to ensure statistical relevance. Apoptotic cells are Annexin V bright and Propidium Iodide (PI) low, late apoptotic cells or necrotic cells are Annexin V and PI bright [37 (link)].
8
Annexin V Apoptosis Assay
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Apoptosis was determined via the Annexin V-FITC kit (Trevigen, Gaithersburg, MD). Briefly, 3 × 105 cells were transfected in a 6-well plate; 24 h later, cells were trypsinized (2 min), washed and centrifuged at 300 × g for 5 min at room temperature. Cells were stained with both Annexin V-FITC and propidium iodide for 15 min in the dark at room temperature. Cells were washed and processed by flow cytometry. Data represent mean ± SD, n = 3 samples/genotype from 3 independent experiments.
9
Propranolol Modulates Apoptosis in HemECs
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HemECs were cultured overnight at 37°C and then assorted into six groups, including the blank control group, dosing+transfection group, transfection group, dosing+inhibitor group, inhibitor group and dosing group. Blank control group, HemECs untreated; Dosing Group, HemECs treated with Propranolol 100 µM/l; Inhibitor group, HemECs treated with PFT-alpha (10 µM/l); Dosing + Inhibitor Group, HemECs treated with PFT-α (10 µM/l) for 3 h and then treated with propranolol (100 µM/l) for 3 h; Transfection Group, high p53 expression model of HemECs untreated; and Dosing + Transfection group, high p53 expression model of HemECs treated with propranolol (100 µM/l) for 3 h. All treatments were at 37°C. The Dosing and transfection group were incubated with propranolol (100 µM/l) for 3 h following transfection. In addition, HemECs in Dosing + Inhibitor group were treated with propranolol for 3 h following addition of PFT-α (10 µm). Following this treatment, cells were collected, washed and subjected to apoptosis analysis using an Annexin V-FITC kit (Trevigen, Inc.), according to the manufacturer's protocol. The cells were analyzed on a FACScan flow cytometer with Cell Quest software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).
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