Fenofibrate, palmitic acid, propidium iodide and RNase A were purchased from Sigma Chemical Company (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit was purchased from BioVision (Mountain view, CA, USA). Antibodies to various proteins were obtained from the following sources: β-Actin was from Sigma Chemical Company. Bax, caspase-3, Cdk1, Cdk2, Cdk4, cyclin B and p21 were purchased from BD Biosciences (San Diego, CA, USA). Cyclin D1, MLKL(pS358) and RIP3(pS227) were purchased from Abcam (Cambridge, MA, USA). ACOT8, Bcl-2, caspase-8, CPT1A, CPT2, cyclin A, cyclin E, FABP1, FASN, PPT1, RIP1 and RIP3 were from GeneTex Inc (Irvine, CA, USA). Caspase-9 and RIP1(pS166) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and -mouse IgG were from Abcam.
Annexin 5 fitc apoptosis detection kit
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Australia
The Annexin V-FITC Apoptosis Detection Kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death. It contains Annexin V conjugated to the fluorescent dye FITC, which binds to phosphatidylserine, a molecule exposed on the surface of apoptotic cells. The kit also includes propidium iodide, a dye that stains the nuclei of late apoptotic or necrotic cells.
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Lab products found in correlation
Facscalibur, by BD (33 mentions)
Facscalibur flow cytometer, by BD (25 mentions)
Acea novocyte flow cytometer, by Agilent Technologies (15 mentions)
Acea novoexpress software, by Agilent Technologies (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Facscanto 2, by BD (9 mentions)
Facscan, by BD (8 mentions)
Facsaria, by BD (7 mentions)
Cellquest software, by BD (7 mentions)
Trypsin edta, by Thermo Fisher Scientific (7 mentions)
Accuri c6 flow cytometer, by BD (7 mentions)
Facscan flow cytometer, by Beckman Coulter (6 mentions)
Facscan flow cytometer, by BD (6 mentions)
Annexin 5 fitc, by Abcam (6 mentions)
Rnase a, by Merck Group (5 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (5 mentions)
Gallios flow cytometer, by Beckman Coulter (5 mentions)
Propidium iodide, by Merck Group (5 mentions)
Flow cytometry, by BD (5 mentions)
Acea novocyte, by Agilent Technologies (5 mentions)
414 protocols using annexin 5 fitc apoptosis detection kit
1
Apoptosis and Cell Cycle Regulation Assay
2
Annexin V-FITC Apoptosis Assay with TRAIL
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Apoptosis was assessed using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA, USA) and PI. Each caspase inhibitor (20 μM), or the same volume of DMSO as a vehicle control, was added 1 h before the addition of TRAIL. After staining with annexin V-FITC/PI, flow cytometric analysis was performed. Analysis was performed using a FACSCalibur flow cytometer.
3
Cell Proliferation and Apoptosis Assay
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Before cell expansion, PL3 cells were labeled with CellVue® Claret (Sigma–Aldrich) at 2 × 10−6 M for 5 min according to the manufacturer's protocol. After 8 days, expanded cells were collected and measured using a BD FACS Calibur™ flow cytometer (dual laser) (BD Biosciences, San Jose, CA). Twenty thousand events of each sample were collected using CellQuest Pro software (BD Biosciences) and cell proliferation index was analyzed by ModFit LT™ version 3.1 (Verity Software House, Topsham, ME).
Apoptosis of expanded cells was detected using Annexin V-FITC Apoptosis Detection Kit (BioVision Inc., Milpitas, CA). Briefly, 2 × 105 detached cells from each group (n = 3) were labeled with FITC conjugated annexin V and propidium iodide for 15 min. Samples were measured using FACS Calibur (BD Biosciences) and analyzed using the FCS Express software package (De Novo Software, Los Angeles, CA).
Expanded cells were incubated with 1 mM hydrogen peroxide (H2O2) at 37 °C for 1 h. To measure intracellular reactive oxygen species (ROS), cells were incubated with 1 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Life Technologies) for 15 min. The plates were read on a FlUOstar OPTIMA (BMG Labtech Inc., Cary, NC) with an excitation wavelength of 485 nm and emission of 530 nm. Samples were assayed in triplicate.
Apoptosis of expanded cells was detected using Annexin V-FITC Apoptosis Detection Kit (BioVision Inc., Milpitas, CA). Briefly, 2 × 105 detached cells from each group (n = 3) were labeled with FITC conjugated annexin V and propidium iodide for 15 min. Samples were measured using FACS Calibur (BD Biosciences) and analyzed using the FCS Express software package (De Novo Software, Los Angeles, CA).
Expanded cells were incubated with 1 mM hydrogen peroxide (H2O2) at 37 °C for 1 h. To measure intracellular reactive oxygen species (ROS), cells were incubated with 1 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Life Technologies) for 15 min. The plates were read on a FlUOstar OPTIMA (BMG Labtech Inc., Cary, NC) with an excitation wavelength of 485 nm and emission of 530 nm. Samples were assayed in triplicate.
4
Cytometric Analysis of Cell Markers
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Apoptosis was measured using an annexin V‐FITC Apoptosis Detection Kit (BioVision). To examine the expression of human EGFR on cancer cells, cells were stained with either FITC‐conjugated anti‐EGFR Ab (NeoMarkers) or isotype‐matched control Ab (Beckman Coulter). To examine the expression of human epidermal growth factor receptor 2 (HER2) on cancer cells, cells were stained with either FITC‐conjugated anti‐HER2 Ab (BD Bioscience) or isotype‐matched FITC‐conjugated mouse IgG (Beckman Coulter). To examine the expression of TRAIL on natural killer (NK) cells, cells were stained with anti‐CD56‐allophycocyanin (APC) Ab (EXBIO) and anti‐TRAIL‐phycoerythrin (PE) Abs. To examine the expression of TRAIL on in vitro expanded T cells, these cells were stained with PE‐conjugated anti‐TRAIL Ab. To determine the subset of T cells, expanded T cells were stained with anti‐CD4‐PE and anti‐CD8‐APC Abs. All analyses were undertaken by flow cytometry (FACSCalibur; Becton Dickinson).
5
Apoptosis Detection by Annexin V-FITC
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For detection of apoptosis, Annexin V-FITC Apoptosis Detection kit (Biovision, Milpitas, CA, USA) was used according to the manufacturer's instructions as we have previously described [42 (link)]. Samples were analyzed using LSR II flow cytometer (BD Bioscience). The sum values of early apoptotic cells (Annexin V+/PI-) and late apoptotic cells (Annexin V+/PI+) represented the total apoptosis.
6
Quantifying Apoptosis in NB4 Cells
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Apoptosis in NB4 cells was quantified by the presence of phosphatidylserine on the outer side of the membrane using the Annexin V-FITC Apoptosis Detection Kit (BioVision). After treatments with esculetin, 2.5 × 105 cells were sedimented at 1200 rpm for 5 minutes and incubated with 500 μl 1x annexin Binding Buffer and 1 μl annexin-FITC for 5 min at room temperature in the dark. Then, 10 μl of PI was added and apoptotic cells were quantified by fluorescence by flow cytometry as indicated before. The results were analyzed using the WinMDI 2.8 software.
Lower left quadrant (FITC−/PI−) corresponds to living cells, lower right quadrant (FITC+/PI−) corresponds to early apoptotic cells, upper right quadrant (FITC+/PI+) corresponds to late apoptotic cells, and upper left quadrant (FITC−/PI+) corresponds to necrotic cells. Quadrants were defined based on the major population of live control cells (low PI; low annexin), and their position kept constant for all other samples.
Lower left quadrant (FITC−/PI−) corresponds to living cells, lower right quadrant (FITC+/PI−) corresponds to early apoptotic cells, upper right quadrant (FITC+/PI+) corresponds to late apoptotic cells, and upper left quadrant (FITC−/PI+) corresponds to necrotic cells. Quadrants were defined based on the major population of live control cells (low PI; low annexin), and their position kept constant for all other samples.
7
Apoptosis and Cell Cycle Analysis
8
Evaluating Cell Apoptosis and Invasion
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The cell apoptosis was evaluated by flow cytometry analysis. Briefly, after treatment with miR-340 mimics, inhibitor or the NC for 48h, the cells were collected and subjected to an annexin V/PI stain using an annexin V-FITC Apoptosis Detection Kit (BioVision, Palo Alto, CA). Then the fluorescence was measured by flow cytometry using a FACS flow cytometer (Becton Dickinson, San Jose, CA). Cell invasion abilities were examined using a 24-well matrigel-coated transwell inserts (BD Biosciences, Bedford, MA, USA). Cells with treatment were transferred on the top of matrigel-coated invasion chambers with serum-free DMEM and were fixed in methanol and stained with 0.1% crystal violet later. The average numbers of cells per field were determined by counting the cells in four random fields per well. All tests were performed in triplicate.
9
Apoptosis Evaluation in HepG2 and Huh7 Cells
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HepG2 and Huh7 cells cultured in six-well plates were treated with OCA for 48 hours. Then, the cells were fixed in cold ethanol for half an hour and stained with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) using an Annexin V‑FITC Apoptosis Detection Kit (BioVision, CA, USA). The cells were then placed at room temperature for 15 min in the dark then analyzed by a FACScan Flow Cytometer (Beckman Coulter, CA, USA). Apoptosis was evaluated in terms of the FITC-positive cells.
10
Annexin V-FITC Apoptosis Assay by Flow
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An Annexin V-FITC apoptosis detection kit (Biovision, Milpitas, CA, USA) was used to detect cell apoptosis. Cells were collected and resuspended in binding buffer, and Annexin V-FITC and propidium iodide were added to each sample and incubated in the dark for 15 min. Annexin V-FITC binding was determined by flow cytometry using FITC signal detector (FL1) and propidium staining by the phycoerythrin emission signal detector (FL2).
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