Apoptosis was measured based on flow cytometry by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining using the FITC Annexin V Apoptosis Detection Kit (BestBio, Shanghai, China), according to the manufacturer's instructions. Flow cytometry was carried out using MoFlo XDP system (Beckman Coulter, Pasadena, CA). Data were analyzed by FlowJo software (Treestar Inc). In co-culture experiments, MV4-11 cells were seed onto the BMSCs monolayer in 6-well plates, then cells were treated with or without αvβ3 blocking antibody (1μg/ml) for 2 hours, sorafenib was then added and cultured for 24 hours. After that, suspension cells were removed by gently shaking and used for apoptosis assay.
Annexin 5 fitc apoptosis detection kit
Manufactured by BestBio
Sourced in China, United States
The Annexin V-FITC Apoptosis Detection Kit is a laboratory tool used for the detection and quantification of apoptosis, a type of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule that translocates to the outer cell membrane during apoptosis. The Annexin V is conjugated to the fluorescent dye FITC, allowing for the visualization and analysis of apoptotic cells through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Lsr flow cytometer, by BD (4 mentions)
Cell cycle detection kit, by BestBio (4 mentions)
Facscan, by BD (3 mentions)
Moflo xdp cell sorter, by Beckman Coulter (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Cellquest software, by BD (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Flow cytometry, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Moflo xdp system, by Beckman Coulter (1 mentions)
Facscalibur fcm, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Poly adp ribose polymerase 1 parp 1, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Z vad fmk, by Selleck Chemicals (1 mentions)
Enhanced chemiluminescence ecl kit, by Thermo Fisher Scientific (1 mentions)
Cytochrome c, by Santa Cruz Biotechnology (1 mentions)
Caspase 9, by Santa Cruz Biotechnology (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (1 mentions)
59 protocols using annexin 5 fitc apoptosis detection kit
1
Annexin V-FITC and PI Apoptosis Assay
2
Apoptosis and Cell Cycle Analysis of GC Cells
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To evaluate apoptosis, the GC cells were collected 48 h after transfection and double stained with Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI; Beyotime, Shanghai, China) using an FITC Annexin V Apoptosis Detection kit (BestBio, Shanghai, China). For the cell cycle assay, GC cells were collected 72 h after transfection. These cells were fixed with 75% ethanol overnight and then stained with PI at 37 °C for 30 min. The harvested cells were then analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA, USA) based on the protocol provided by manufacturer.
3
Quantification of Apoptosis by Flow Cytometry
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The level of Apoptosis was quantified by FITC-Annexin V apoptosis detection kit (BestBio, China). Firstly, AML-12 cells were washed by cold PBS three times. Then cells sedimentation were resuspended in binding buffer at a density of 1 × 106/ml. Next, adding 10 μl PI and 5 μl Annexin V-FITC to stain apoptosis cells. Staining cells were calculated with BD LSR flow cytometer (BD Biosciences, San Jose, CA, United States) and the data was analyzed by a software named FlowJo (Gondhalekar et al., 2018 (link); Libregts et al., 2018 (link)).
4
Evaluating Apoptosis in Hepatocytes
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To assess apoptosis of hepatocytes in the liver tissues, terminal deoxyuridine nick end labeling (TUNEL) staining was performed using a kit (Roche, Switzerland) according to the manufacturer’s instructions. Briefly, sections of the liver from six rats of each of the 24 h groups (control, burn, and burn + carnitine) were stained for apoptotic cells, and 10 randomly selected fields of each section were photographed to count the TUNEL-positive cells. The data were quantified as the percentage of apoptotic cells per hundred hepatocytes.
To assess apoptotic cells in vitro, the hepatocyte cell line HepG2 was cultured in a 6-well plate at a density of 4 × 105 cells/well and incubated overnight at 37 °C. After 24 h of treatment (as indicated in the section below), the cells were collected and stained using the FITC Annexin V Apoptosis detection kit (Bestbio, Shanghai, China). Apoptotic HepG2 cells were analyzed via flow cytometry (FACS Calibur FCM; Becton–Dickinson, San Jose, CA, USA).
To assess apoptotic cells in vitro, the hepatocyte cell line HepG2 was cultured in a 6-well plate at a density of 4 × 105 cells/well and incubated overnight at 37 °C. After 24 h of treatment (as indicated in the section below), the cells were collected and stained using the FITC Annexin V Apoptosis detection kit (Bestbio, Shanghai, China). Apoptotic HepG2 cells were analyzed via flow cytometry (FACS Calibur FCM; Becton–Dickinson, San Jose, CA, USA).
5
Annexin V Apoptosis Assay
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The different groups of EC9706 and EC1 cells were harvested and resuspended at 107 cells/mL in 1× binding buffer. After double staining with Fluorescein-5-isothiocyanate (FITC)-Annexin V and propidium iodide of FITC-Annexin V Apoptosis Detection Kit (BestBio), cells were analyzed using FACScan flow cytometer (BD Biosciences) equipped with Cell Quest software (BD Biosciences).
6
Annexin V-FITC Apoptosis Assay
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Cells were harvested and apoptosis was detected by a FITC Annexin V Apoptosis Detection Kit (BB-4101-3, BestBio, Nanjing, China). According to the manufacturer’s protocols, cells were suspended in 400 µL 1× binding buffer with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI) followed by incubation for 15 min at 4 °C in the dark. Finally, apoptosis was analyzed by flow cytometry (Thermo Fisher Scientific, Attune NxT, Waltham, MA, USA).
7
Apoptosis Induction by Oxidative Stress
8
Cell Cycle and Apoptosis Analysis
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The GC cells were collected 48 h after transfection, and the cell cycle and apoptosis were evaluated using the Cell-cycle Detection Kit and FITC Annexin V Apoptosis Detection kit (BestBio, Shanghai, China) respectively. The harvested cells were then analyzed by flow cytometry (FACScan, BD Biosciences).
9
Apoptosis and Necrosis Quantification
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To distinguish living cells, apoptotic cells and necrotic cells accurately and count the proportion of each group of cells, it was assayed using FITC Annexin V Apoptosis Detection Kit (Bestbio, Shanghai, China). Briefly, the treated cells for 48 h were digested by trypsin without EDTA and washed with PBS for 3 times strictly. After Annexin V-FITC and PI staining solution were added to the suspended cells, the reaction was incubated in the dark place for 15 min, followed by sample loading and detection through flow cytometry.
10
Apoptosis Detection in Drug-Treated Cells
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Apoptosis was detected through flow cytometry using FITC Annexin V Apoptosis Detection Kit (Bestbio, Shanghai, China). Drug-treated cells (culture in the incubator for 24 h) were digested by trypsin without EDTA, centrifuged, and resuspended with PBS for 3 times strictly. The fluorescence maker was added and cells were incubated in a dark place at 2-8°C for 15 min, followed by sample loading and detection through flow cytometry. All samples were analyzed within 1 h to ensure the effect.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was conducted with the TUNEL kit according to the manufacturer’s instructions. In brief, the liver tissue was embedded in paraffin, then deparaffinized with xylene, stained with TUNEL reaction mixture, then stained by DAPI staining and anti-fluorescence quenching were performed. Finally, the obtained slices were observed and photographed at a suitable high magnification, with the apoptotic cells appearing green and the nuclei appearing in blue.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was conducted with the TUNEL kit according to the manufacturer’s instructions. In brief, the liver tissue was embedded in paraffin, then deparaffinized with xylene, stained with TUNEL reaction mixture, then stained by DAPI staining and anti-fluorescence quenching were performed. Finally, the obtained slices were observed and photographed at a suitable high magnification, with the apoptotic cells appearing green and the nuclei appearing in blue.
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