Annexin v 7 aad kit

Manufactured by Beckman Coulter

Sourced in France, United States

The Annexin V/7-AAD kit is a laboratory reagent used to detect and measure apoptosis, a form of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, and 7-AAD, a dye that stains DNA, to identify and distinguish between viable, early apoptotic, and late apoptotic/necrotic cells through flow cytometry analysis.

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Lab products found in correlation

Coulter epics xl, by Beckman Coulter (1 mentions) 7 aminoactinomycin d, by Beckman Coulter (1 mentions) 12 well plate, by Sarstedt (1 mentions) Protease inhibitor tablet, by Thermo Fisher Scientific (1 mentions) 4 hba, by Merck Group (1 mentions) γ 32p atp, by MP Biomedicals (1 mentions) 2 4 6 thba, by Merck Group (1 mentions) 3 4 5 thba, by Merck Group (1 mentions) Qpcr reagents, by Thermo Fisher Scientific (1 mentions) 3 4 dhba, by Merck Group (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Trypsin edta, by Merck Group (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Vybrant dyecycle green stain, by Thermo Fisher Scientific (1 mentions) Cytoflex flow cytometer, by Beckman Coulter (1 mentions)

7 protocols using annexin v 7 aad kit

Annexin V and 7-AAD Apoptosis Assay

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Annexin V/7-AAD Staining. Second generation of spheroids was collected and centrifuged and pellets were trypsinized and washed twice with PBS. The cells were stained with FITC labeled annexin V/7-AAD (7-aminoactinomycine-D) according to the manufacturer's indication (annexin V/7-AAD kit; Beckman Coulter, Marseille, France). In particular, a washed cell pellet (5 × 10⁴ cells/mL) was resuspended in 500 μL binding buffer; 10 μL of annexin V together with 20 μL 7-AAD was added to 470 μL cell suspension. The cells were incubated for 15 min on ice in the dark. Apoptosis assay was performed three times.
ALDH, cell cycle, and apoptosis were evaluated by flow cytometry using an EPICS Coulter XL (Beckman-Coulter Inc.). Data were analyzed by Modfit LT Software (Veruty Software Inc., USA).

Pisanu M.E., Noto A., De Vitis C., Masiello M.G., Coluccia P., Proietti S., Giovagnoli M.R., Ricci A., Giarnieri E., Cucina A., Ciliberto G., Bizzarri M, & Mancini R. (2014). Lung Cancer Stem Cell Lose Their Stemness Default State after Exposure to Microgravity. BioMed Research International, 2014, 470253.

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Annexin V-FITC/7-AAD Apoptosis Assay

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Cell clumps were collected and centrifuged, and pellets were trypsinized and washed twice with PBS. Adherent cells and ground control cells were trypsinized and washed twice with PBS. The cells were stained with FITC-labeled annexin V/7-AAD (7-aminoactinomycin-D) according to the manufacturer’s instructions (annexin-V/7-AAD kit; Beckman Coulter, Marseille, France). Briefly, a washed cell pellet (5 × 104 cells/mL) was resuspended in 500 μL binding buffer; 10 μL of annexin-V together with 20 μL 7-AAD was added to 470 μL cell suspension. The cells were incubated for 15 min on ice in the dark. The samples were analyzed by flow cytometry. Apoptosis assay was performed three times.

Monti N., Masiello M.G., Proietti S., Catizone A., Ricci G., Harrath A.H., Alwasel S.H., Cucina A, & Bizzarri M. (2021). Survival Pathways Are Differently Affected by Microgravity in Normal and Cancerous Breast Cells. International Journal of Molecular Sciences, 22(2), 862.

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Quantifying Apoptosis via Annexin V/7-AAD

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Cell apoptosis was assessed using the Annexin V/7-AAD kit (#IM3614; Beckman Coulter) according to the manufacturer’s protocol. 100,000 BxPC3, Sw1990, and C-PDX P4604 cells were grown in 1 ml of medium/10% FCS in 12-well plates (Sarstedt) at 37°C for 24 h, before serum starvation in culture medium/2% FCS for 24 h. After medium removal, cells were incubated with various concentrations of BiXAb™ or 2MAbs in medium/2% FCS at 37°C for 48 h. Some cells were incubated with staurosporine (200 nM; positive control for apoptosis) at 37°C for 3 h. After centrifugation, the cell pellet of each well was labeled with 10 µl of annexin-FITC and 20 µl of 7-AAD. After 15 min incubation at 4°C in the dark, Annexin V/7-AAD labeling was measured with a Gallios flow cytometer and analyzed with the Kaluza software.

Rabia E., Garambois V., Dhommée C., Larbouret C., Lajoie L., Buscail Y., Jimenez-Dominguez G., Choblet-Thery S., Liaudet-Coopman E., Cerutti M., Jarlier M., Ravel P., Gros L., Pirot N., Thibault G., Zhukovsky E.A., Gérard P.E., Pèlegrin A., Colinge J, & Chardès T. (2023). Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer. Frontiers in Immunology, 14, 1168444.

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Biochemical Assays and Reagents

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2,4,6-THBA, 3,4-DHBA, 3,4,5-THBA, 4-HBA and trypsin-EDTA were obtained from Sigma Aldrich (St. Louis, MO, USA); H1 Histones and Immobilon membranes from EMD Millipore (Billerica, MA, USA); ³²P γ-ATP from MP Biochemicals (Solon, OH, USA); RT-PCR reagents from New England Biolabs (NEB, Ipswich, MA, USA); qPCR reagents from Applied Biosystems (Foster City, CA, USA); Annexin V/7-AAD kit from Beckman Coulter (Miami, FL, USA); Super Signal™ West Pico Chemiluminescent Substrate, protease inhibitor tablets and all other chemicals were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Sankaranarayanan R., Valiveti C.K., Kumar D.R., Van slambrouck S., Kesharwani S.S., Seefeldt T., Scaria J., Tummala H, & Bhat G.J. (2019). The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention. Cancers, 11(3), 427.

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Quantifying Early Apoptosis by Flow Cytometry

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Cells were collected and washed three times with ice-cold PBS. Early phase apoptosis was determined by the Annexin-V/7AAD Kit (PN IM3614, Beckman Coulter) using a FACSCalibur flow cytometer (Becton and Dickinson, San Jose, CA) using the manufacturer’s protocol. Briefly, cells were washed and resuspended in binding buffer. Annexin-V (1:10) and 7-Amino-Actinomycin (7AAD, 1:20) were added and incubated for 15 min on ice in the dark. Binding buffer was added and analyzed by flow cytometry followed within 1 hr. Annexin-V-positive and 7AAD-positive cells were considered as apoptotic cells.

Jansen G., Al M., Assaraf Y.G., Kammerer S., van Meerloo J., Ossenkoppele G.J., Cloos J, & Peters G.J. (2023). Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells. Cancer Drug Resistance, 6(3), 430-446.

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Flavonoid-Induced Apoptosis and Cell Cycle Analysis

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Cells were treated for 72 h with 100 µM nobiletin and 5,6,7,3′,4′,5′-hexamethoxyflavone. Floating cells and trypsinized adherent cells were combined and washed with cold PBS or cell culture medium. For detection of apoptosis, cells were stained with an Annexin V/7-AAD kit (Beckman Coulter, Miami, FL, USA) using the manufacturer's protocol. In brief, cells were incubated with Annexin V and 7-AAD in ice-cold binding buffer in the dark. After 15 min the samples were mixed with more binding buffer and analyzed within 30 min. Positive controls for apoptosis induction included Hs578T and Hs578Ts(i)₈ cells treated with 10 µM camptothecin for 72 h. Cell cycle analysis was investigated by adding Vybrant^® DyeCycle™ Green Stain (Thermo Fisher Scientific) to 1 ml cell suspension at a final concentration of 250 nM. After 30-min incubation at 37°C, the samples were analyzed by flow cytometry and compared against DMSO-treated cells. All these experiments were performed on a CytoFLEX flow cytometer (Beckman Coulter) using CytExpert 2.0 software.

Borah N., Gunawardana S., Torres H., McDonnell S, & Van Slambrouck S. (2017). 5,6,7,3′,4′,5′-Hexamethoxyflavone inhibits growth of triple-negative breast cancer cells via suppression of MAPK and Akt signaling pathways and arresting cell cycle. International Journal of Oncology, 51(6), 1685-1693.

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Apoptosis Analysis by Flow Cytometry

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Cell clumps were collected and centrifuged and pellets were trypsinized and washed twice with PBS. Adherent cells and ground control cells were trypsinized and washed twice with PBS. The cells were stained with FITC labeled annexin V/7-AAD (7-aminoactinomycin-D) according to the manufacturer's instructions (annexin V/7-AAD kit; Beckman Coulter, Marseille, France). Briefly, a washed cell pellet (5 × 10⁴ cells/mL) was resuspended in 500 μL binding buffer; 10 μL of annexin V together with 20 μL 7-AAD was added to 470 μL cell suspension. The cells were incubated for 15 min on ice in the dark. The samples were analyzed by flow cytometry. Apoptosis assay was performed three times.

Masiello M.G., Cucina A., Proietti S., Palombo A., Coluccia P., D'Anselmi F., Dinicola S., Pasqualato A., Morini V, & Bizzarri M. (2014). Phenotypic Switch Induced by Simulated Microgravity on MDA-MB-231 Breast Cancer Cells. BioMed Research International, 2014, 652434.

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