Cell lines were seeded in six-well plates at a density of 0.6 × 105 cells/mL and were allowed to attach overnight. The following day, cells were treated with the concentration of 5-FU corresponding to the IC50 value determined for AGS and Kato III wild-type cell lines. Cells treated only with dimethyl sulfoxide (DMSO, Applichem) were used as control. After 48 h, cells were collected by trypsinization and pellets were resuspended in Binding Buffer 4x (annexin V Apoptosis detection Kit, eBioscience, Affymetrix, Santa Clara, CA, USA). Each sample was first stained with annexin V Alexa Fluor 594 conjugate (Invitrogen, Carlsbad, CA, USA) or annexin V APC (ImmunoTools, Germany) and then with propidium iodide (PI) (annexin V Apoptosis detection Kit, eBioscience, Affymetrix, Santa Clara, CA, USA). The percentage of apoptosis was measured using the FACS Aria II flow cytometer. The results were analyzed using FlowJo software (version 7.6.1).
Annexin 5 apoptosis detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Italy
The Annexin V Apoptosis Detection Kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death. The kit provides reagents and protocols to label and identify cells undergoing apoptosis. The core function of the kit is to facilitate the measurement and analysis of apoptosis through the detection of phosphatidylserine exposure on the cell surface, a hallmark of the apoptotic process.
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Lab products found in correlation
Facscalibur, by BD (12 mentions)
Lsrfortessa, by BD (10 mentions)
Facscanto 2, by BD (6 mentions)
Cytofix cytoperm kit, by BD (6 mentions)
Cytoflex, by Beckman Coulter (5 mentions)
Ifn γ, by Thermo Fisher Scientific (5 mentions)
Celltrace violet, by Thermo Fisher Scientific (5 mentions)
Propidium iodide, by Thermo Fisher Scientific (4 mentions)
Lsr 2 flow cytometer, by BD (4 mentions)
Software, by FlowJo (4 mentions)
Flow cytometry, by BD (4 mentions)
Facscalibur cytometer, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
Facsaria, by BD (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Bortezomib, by LC Laboratories (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Facsdiva software, by BD (3 mentions)
242 protocols using annexin 5 apoptosis detection kit
1
Apoptosis Induction by 5-FU in Gastric Cancer Cells
2
Annexin V-FITC Apoptosis Assay
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After dissociation by 0.25% trypsin-EDTA and harvesting by centrifugation, the cells were washed thrice with phosphate-buffered saline (PBS). An Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific, USA) was used to determine apoptosis in accordance with the manufacturer's instructions. Briefly, the cells were resuspended in 100 μL binding buffer containing 5 μL of FITC-conjugated Annexin V antibody, and incubated for 15 min at room temperature. Then, they were washed and resuspended in a 200 μL binding buffer with 5 μL of propidium iodide (PI). Finally, the cells were analyzed using the CytoFlex (Beckman Coulter) flow cytometry and FlowJo software (Treestar).
3
Apoptosis and Necroptosis Pathway Assays
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DHA (D2534) and EPA (E2011) were purchased from Sigma. DHA and EPA were dissolved in ethanol to produce a 100 mM stock solution. Annexin V apoptosis detection kit (88-8005-74) was obtained from Thermo Fisher. Bortezomib was obtained from LC laboratories and dissolved in DMSO. Pan-caspase inhibitor Z-VAD-FMK (tlrl-vad) was purchased from In vivoGen. Caspase-3 inhibitor Z-DEVD-FMK (FMK004), caspase-8 inhibitor Z-IETD-FMK (FMK007), caspase-10 inhibitor Z-AEVD-FMK (FMK009) and MG132 (1748) were obtained from R&D systems. Bis(sulfosuccinimidyl) suberate (BS3) (21586) were obtained from Thermo Scientific Pierce Biotechnology. Anti-Flag M2 Magnetic Beads was obtained from Sigma-Aldrich. Antibodies for MLKL (#14993), RIPK1 (#3493), RIPK3 (#13526), phospho-Ser358-MLKL (#91689), phospho-Ser166-RIPK1 (#65746), phospho-Ser227-RIPK3 (#93654), cleaved-caspase3 (#9661) and Flag M2 antibody (#2368) were purchased from Cell Signaling Technology; anti-GAPDH (sc-47724) was obtained from Santa Cruz Biotechnology. Anti-MLKL antibody (GTX107538) for immunoprecipitation was obtained from GeneTex (Hsinchu, Taiwan). Horse radish peroxidase-conjugated secondary antibodies were purchased from Dako Agilent.
4
Flow Cytometric Analysis of Lung Leukocytes
5
Annexin V Apoptosis Assay by Flow Cytometry
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The percentages of apoptotic cells in each group were determined by flow cytometry using the Annexin V Apoptosis Detection Kit (88‐8007; Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the different groups of cells (105/tube) were stained in duplicate with 5 μL annexin V‐allophycocyanin and 5 μL 7‐AAD solutions for 15 minutes at room temperature. After being washed, the percentages of apoptotic cells were determined by flow cytometry and analyzed using the FlowJo (FlowJo LLC, Ashland, OR, USA) software.
6
Osteoblast Apoptosis Assessment
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Osteoblasts were seeded into 6-well plates (1×105 cells/well) and cultured in DMEM at 37°C in 5% CO2 for 48 h and treated as aforementioned. Cells were harvested with 0.25% trypsin (Sigma-Aldrich; Merck KGaA) at 37°C in 5% CO2 for 20 min for apoptotic analysis using the Annexin V apoptosis detection kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were stained with Annexin V and PI for 30 min in the dark at room temperature followed by BD FACS Calibur™ flow cytometry analysis using CellQuest Pro 5.1 software (BD Biosciences). Annexin V positive and PI negative stained cells indicated early apoptosis.
7
Evaluating Combination Therapies for Vemurafenib-Resistant Thyroid Cancer
8
Annexin V-Based Apoptosis Detection
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An annexin V apoptosis detection kit (Thermo Fisher Scientific, Inc.) was used for the analysis of apoptosis. Following transfection, the cells were trypsinized, harvested and resuspended. A total of 2×105 cells were harvested and washed twice with cold PBS, and subsequently resuspended in 500 µl binding buffer. A total of 10 µl annexin V-fluorescein isothiocyanate and 10 µl propidium iodide was added to the solution and mixed well. Following 15 min of incubation, the cells were analyzed by flow cytometric analysis (BD Biosciences, San Jose, CA, USA) with FCS Express 6 flow cytometry software (FCS Express 6.04.0015, De Novo Software, Los Angeles, CA, USA).
9
Evaluating HATMSC2-MVs Impact on Cell Viability
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In order to determine the effect of HATMSC2-MVs on the viability of the cells, an Annexin V Apoptosis Detection Kit (Thermo Scientific, Carlsbad, CA, USA) was used. ES-2 and OAW-42 cells were seeded in a 24-well plate at a density of 25 × 103 in the DMEM + 10% FBS and OptiMEM GlutaMax + 3% FBS media (mixed in equal proportions). Before the analysis, the cells were treated with MVs at a ratio of 1:1 (1 MV per cell), 5:1 (5 MVs per cell), 10:1 (10 MVs per cell), and 100:1 (100 MVs per cell) for 72 h. ES-2 and OAW-42 cells without MVs were used as a negative control. After incubation with MVs, the cells were stained with Annexin V and propidium iodide according to the manufacturer’s recommendations. The cells were analyzed for live (Annexin V negative and propidium iodide negative), early apoptotic (Annexin V positive and propidium iodide negative), late apoptotic (Annexin V positive and propidium iodide positive), and necrotic cells (Annexin V negative and propidium iodide positive) using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis was performed using Flowing Software 2 (Perttu Terho, Turku Centre for Biotechnology, Finland).
10
Quantifying Apoptosis in Cancer Cells
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Annexin V stained flipped phospholipids on the HCT 116 and HepG2 cell membranes and used in determination of apoptosis after treatment with Rc, Ro, ML, MLn at 24 h of drug incubation. Annexin V apoptosis detection kit purchased from (ThermoFisher Scientific). The Annexin V-based apoptosis of the treated and untreated HCT 116 and HepG2 cancer cells with the IC50s of these formulations were analyzed in triplicate using the fluorescence module of the BMG LABTECH, Germany.
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