Annexin 5 binding

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Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). Annexin V binding is commonly used to detect and quantify cells undergoing apoptosis, as PS is translocated from the inner to the outer leaflet of the plasma membrane during this process.

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Lab products found in correlation

Propidium iodide, by BD (3 mentions) Facscalibur, by BD (2 mentions) Cytotox glo reagent, by Promega (2 mentions) White 96 well plate, by Corning (2 mentions) Cytoflex, by Beckman Coulter (2 mentions) Cytexpert software, by Beckman Coulter (2 mentions) Annexin 5 fitc, by BD (2 mentions) Automated cell counter, by Bio-Rad (1 mentions) Facsvantage se, by BD (1 mentions) Apc conjugated annexin 5, by BD (1 mentions) 7 aad, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Celltiter 96 aqueous one solution reagent, by Promega (1 mentions) 7 aminoactinomycin d 7 aad expression, by Thermo Fisher Scientific (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Promega (1 mentions) Dmem high glucose, by Merck Group (1 mentions) Z vad fmk, by R&D Systems (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Annexin V binding assay kit 1 × Annexin V-binding buffer solution Annexin V-FITC binding solution Annexin V-FITC binding buffer Annexin V binding buffer (pH 7.4) Annexin V–FITC binding assay Annexin V-fluorescein isothiocyanate (FITC)-binding assay Annexin V binding buffer Annexin V binding buffer 1×, Annexin V binding solution

9 protocols using annexin 5 binding

Annexin V-PI Apoptosis Assay

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Cells were seeded in a six-well plate and treated through various treatments to modulate MCL1 or p73 expression as they were in previous experiments. After the 24 or 48 h treatment, cells were collected through trypsin dissociation and washed twice with sterile DPBS. Cell number was counted on a BioRad Automated Cell Counter. After final wash, cells were pelleted at 1000×g for 10 min. The cell pellet was resuspended in 1× Annexin V binding buffer (BD Pharmingen, 556454) and 5 × 10⁵ cells were transferred to a FACS tube containing 5 μL propidium iodide (PI) staining solution (BD Pharmingen, 51-66211E) and 5 μL FITC-Annexin V (BD Pharmingen, 556419). Cells were incubated for 15 min in the dark. FACS was collected on a BD LSRFortessa and analyzed using FlowJo v10. Compensation controls used for analysis include unstained cells and cells stained for each individual fluorophore (FITC-Annexin V and Cy5-PI). Gating strategy eliminated cell debris and doublets through forward and side scatter plots.

Widden H., Kaczmarczyk A., Subedi A., Whitaker R.H, & Placzek W.J. (2020). MCL1 binds and negatively regulates the transcriptional function of tumor suppressor p73. Cell Death & Disease, 11(11), 946.

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Annexin V Apoptosis Assay

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Cells were seeded in six-well plates and treated the next day with either the indicated amount of drug, vehicle (DMSO), or combination. Cells were incubated for two days, washed twice with ice-cold PBS, and resuspended in 1X annexin V binding buffer (10mM HEPES, 140mM NaCl, 2.5mM CaCl2; BD Biosciences). Surface exposure of phosphatidylserine was measured using APC-conjugated annexin V (BD Biosciences). 7-AAD (BD Biosciences) was used as a viability probe. Experiments were analyzed at 20,000 counts/sample using BD FACSVantage SE. Gatings were defined using untreated/unstained cells as appropriate.

Anderson G.R., Winter P.S., Lin K.H., Nussbaum D.P., Cakir M., Stein E.M., Soderquist R.S., Crawford L., Leeds J.C., Newcomb R., Stepp P., Yip C., Wardell S.E., Tingley J.P., Ali M., Xu M., Ryan M., McCall S.J., McRee A.J., Counter C.M., Der C.J, & Wood K.C. (2017). A landscape of therapeutic cooperativity in KRAS mutant cancers reveals principles for controlling tumor evolution. Cell reports, 20(4), 999-1015.

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Hematopoietic Stem Cell Immunophenotyping

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Monoclonal antibodies against Mac1, Gr1, B220, and CD3 (all from eBioscience) were used for peripheral blood analysis. Bone marrow lineage staining was carried out with biotinylated antibodies against Gr1, Ter119, B220, and CD3 (all from eBioscience). Bone marrow cells were also stained with antibodies against cKit, Sca1, CD16/32, CD34, FLT3, CD150, CD48, and IL7R (all from eBioscience). Flow cytometry and BrdU staining were carried out as previously described (70 ). Apoptosis was determined by AnnexinV binding (BD Biosciences). Flow cytometry was performed on FACSCalibur or LSRII flow cytometers (BD Biosciences) and analyzed with FlowJo software. Cell sorting was carried out on a FACSAria II (BD Biosciences).

Lim Y., Gondek L., Li L., Wang Q., Ma H., Chang E., Huso D.L., Foerster S., Marchionni L., McGovern K., Watkins D.N., Peacock C.D., Levis M., Smith B.D., Merchant A.A., Small D, & Matsui W. (2015). Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia. Science translational medicine, 7(291), 291ra96.

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MTT Viability, Apoptosis, and Cytotoxicity Assays

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[3–5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] MTT cell viability assays were performed in 96-well dishes using Cell Titer 96 Aqueous One solution reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are expressed as a percentage of mock-infected cells analyzed in parallel.
Alternatively, cells cultured in 24-well plates (2 cm diameter) were treated as indicated in each figure legend (concentration of drug and time). Media and trypsinized adherent cells were centrifuged and analyzed for annexin V binding (BD Bioscience, San Jose, CA, USA) and propidium iodide (BD Bioscience) staining as described by the manufacturer. Cells were analyzed by flow cytometry using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change of mock-infected cells analyzed in parallel.

Fox C.R, & Parks G.D. (2019). Histone Deacetylase Inhibitors Enhance Cell Killing and Block Interferon-Beta Synthesis Elicited by Infection with an Oncolytic Parainfluenza Virus. Viruses, 11(5), 431.

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Apoptosis Analysis of GANT61 and Cyclopamine Treated Cells

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Cells (0.25 × 10⁶) treated with GANT61 or Cyclopamine were incubated in 48-well plates in RPMI-1640 containing 10% FBS for 48 h. Cells were washed with Annexin V binding buffer and stained with 1 μg Annexin V FITC (BD Biosciences) for 20 min at 4°C in the dark. Cells were again washed with Annexin V binding buffer and then stained with 0.5 μg propidium iodide. Cells were immediately processed using a FACSCalibur (BD Biosciences) and data was analyzed using FlowJo software (FlowJo LLC).

Jackson D.A., Smith T.D., Amarsaikhan N., Han W., Neil M.S., Boi S.K., Vrabel A.M., Tolosa E.J., Almada L.L., Fernandez-Zapico M.E, & Elsawa S.F. (2015). Modulation of the IL-6 Receptor α Underlies GLI2-mediated regulation of Immunoglobulin Secretion in Waldenström macroglobulinemia cells. Journal of immunology (Baltimore, Md. : 1950), 195(6), 2908-2916.

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Quantification of Apoptosis and Proliferation

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Apoptosis was quantified using flow cytometry as detailed in our previous publication (37 (link)), for annexin V binding (BD Biosciences) and 7-aminoactinomycin D (7-AAD) expression (eBioscience). Caspase 3 activity was evaluated by immunoblotting. An MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation assay was carried out following the manufacturer’s instructions (Promega).

Wang L., Howell M.E., Sparks-Wallace A., Zhao J., Hensley C.R., Nicksic C.A., Horne S.R., Mohr K.B., Moorman J.P., Yao Z.Q, & Ning S. (2021). The Ubiquitin Sensor and Adaptor Protein p62 Mediates Signal Transduction of a Viral Oncogenic Pathway. mBio, 12(5), e01097-21.

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Annexin V/PI Flow Cytometry Protocol

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Cells cultured in 24-well plates or 48-well plates were treated as indicated in each figure legend. Media and trypsinized adherent cells were centrifuged and analyzed for annexin V binding (BD Bioscience, San Jose, CA, USA) and propidium iodide (BD Bioscience) staining as described by the manufacturer. Cells were analyzed by flow cytometry using the CytoFLEX (Beckman Coulter, Brea, CA, USA) and 10,000 independent events were analyzed using CytExpert software (Beckman Coulter).
Alternatively, cytotoxicity assays were performed in 96-well white plates (Corning, Corning, NY, USA) using CytoTox-Glo reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel. Functional caspase assays were performed in 96-well white plates (Corning) using Caspase-Glo 9, 8, or 3/7 assays (Promega) according to the manufacturer’s instructions. Data are expressed as a fold change over mock-infected cells analyzed in parallel.

Cruz M.A, & Parks G.D. (2020). La Crosse Virus Infection of Human Keratinocytes Leads to Interferon-Dependent Apoptosis of Bystander Non-Infected Cells In Vitro. Viruses, 12(3), 253.

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Neutrophil Apoptosis Assay with ST7 and G-CSF

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Peripheral blood was collected and assays were performed with the approval of the Institutional Review Board of the University of Massachusetts School of Medicine. Normal neutrophils were harvested from healthy volunteers, purified by sedimentation through 6% dextran, placed in culture in HBSS containing 1% human serum albumin serum, and incubated with ST7 at 200 μM, ST7-S at 50 μM, or G-CSF at 100 U/mL, at 37°C in a shaking water bath. Samples were analyzed after 24 and 43 hrs of exposure; apoptotic cell fraction was assayed by Annexin V binding (BD BioSciences; San Jose, CA) and a TUNEL assay using FITC-dUTP (Chemicon; Billerica, MA).

Faller D.V., Castaneda S.A., Zhou D., Vedamony M., Newburger P.E., White G.L., Kosanke S., Plett P.A., Orschell C.M., Boosalis M.S, & Perrine S.P. (2016). An oral Hemokine™, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo. Blood cells, molecules & diseases, 63, 1-8.

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Melanoma Cell Line Characterization

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B16 mouse melanoma cell lines F0 (ATCC CRL-6322), F1 (ATCC CRL-6323), and F10 (ATCC CRL-6475) were purchased from the ATCC. Cell lines were expanded immediately after purchase and frozen in liquid nitrogen (lN2). Cells were routinely cultured in high glucose DMEM (Sigma-Aldrich) supplemented with 8% FBS, utilized within 10 passages after thawing from lN2, and regularly tested for Mycoplasma prior to injection into animals. Low metastatic potential B16F0 cells stably expressing EGFP or mCherry were generated by transfection of the EGFP-N1 vector or mCherry-C1 vector (Takara Bio) using Lipofectamine 2000 (Invitrogen) followed by selection in 1.2 mg/mL G418 (Life Technologies) and two rounds of fluorescenceactivated cell sorting to select stable pools of cells with high fluorescent protein expression (top 10%). In some experiments, B16F0 cells were maintained in normal B16F0 medium containing methotrexate (MTX; Cayman Chemicals) without or with z-vad-fmk (R&D Systems) or media conditioned by either MoDM or monocytes. After these treatments, apoptosis and cell death were quantified by flow cytometry using Annexin V binding (BD Biosciences) and DAPI uptake (Thermo Fisher Scientific).

Arif A.A., Huang Y.H., Freeman S.A., Atif J., Dean P., Lai J.C.Y., Blanchet M.R., Wiegand K.C., McNagny K.M., Underhill T.M., Gold M.R., Johnson P, & Roskelley C.D. (2021). Inflammation-Induced Metastatic Colonization of the Lung Is Facilitated by Hepatocyte Growth Factor-Secreting Monocyte-Derived Macrophages. Molecular cancer research : MCR, 19(12).

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