Annexin 5 solution

Manufactured by BD

Sourced in United States

Annexin V solution is a laboratory reagent used in various cell biology and biochemical applications. It is a recombinant protein that binds to phosphatidylserine, a lipid present on the surface of cells undergoing programmed cell death (apoptosis). The Annexin V solution can be used to detect and quantify apoptotic cells in a sample.

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Lab products found in correlation

Facscalibur flow cytometer, by BD (3 mentions) Annexin binding buffer, by Roche (2 mentions) Propidium iodide, by BD (2 mentions) Lsr fortessa x 20 cell, by BD (1 mentions) Annexin 5, by BD (1 mentions) Propidium iodide, by Merck Group (1 mentions) Tunel test solution, by Beyotime (1 mentions) Propidium iodide solution pi, by BD (1 mentions) Annexin 5 binding buffer 1x, by BD (1 mentions)

Close spelling variants of this product

1 × Annexin V-binding buffer solution Annexin V-FITC and PI solution Annexin V-FITC solution Annexin V-FITC staining solution Annexin V‐FITC/propidium iodide solution Annexin V-FITC binding solution Annexin V/Propidium Iodide (PI) staining solution Annexin V–fluorescein isothiocyanate solution Annexin V binding solution Annexin V

7 protocols using annexin 5 solution

Quantifying Apoptosis via Flow Cytometry

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On 4- or 6 days post-transduction of sgRNA-containing lentiviruses, 30,000 cells were seeded into 24-well plate and harvested after 72 hr. Cells were pelleted down and washed with PBS. For detection of apoptotic cells, the cell pellet was resuspended in 1x Annexin V buffer containing 2.5 μl of Annexin V solution (BD Biosciences) and 2.5 μl of propidium iodide and incubated for 15 min in the dark. The proportion of apoptotic cells were analyzed using the LSR Fortessa X-20 cell analyser (BD Biosciences) and FlowJo (version 10.5.3, BD Biosciences), considering all single- and double-stained cells as apoptotic cells. Gating strategy for detection of apoptotic cells using flow cytometry can be found in Supplementary file 8.

Chai A.W., Yee P.S., Price S., Yee S.M., Lee H.M., Tiong V.K., Gonçalves E., Behan F.M., Bateson J., Gilbert J., Tan A.C., McDermott U., Garnett M.J, & Cheong S.C. (2020). Genome-wide CRISPR screens of oral squamous cell carcinoma reveal fitness genes in the Hippo pathway. eLife, 9, e57761.

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Annexin V-PI Apoptosis Assay for Cell Death

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After treatment at the IC₅₀ + SD concentrations of the respective samples, the cells were trypsinized and the cell pellet was resuspended in 500 µL of ice-cold PBS and centrifuged at 2000 rpm for 5 min at 4 °C. After discarding the supernatant, 100 µL of binding buffer (0.1 M Hepes (pH 7.4), 1.4 M NaCl and 25 mM CaCl₂) was added to the pellet. Then, 5 µL of Annexin V solution (BD, USA) and 10 µL of PI solution (50 µg/mL) were added to each microtube. The microtubes were left for 15 min protected from light and at room temperature. Finally, 300 µL of binding buffer was added. The samples were placed on ice and taken for FACS analysis immediately. Hydrogen peroxide (H₂O₂) is an oxidizing agent that acts directly on the lipid membrane and DNA, leading to membrane rupture and causing cell death by necrosis. This, at a concentration of 29.4 µM, was used as a positive control for the experiment.

Alves R.C., Perosa Fernandes R., Lira de Farias R., da Silva P.B., Santos Faria R., Quijia C.R., Galvão Frem R.C., Azevedo R.B, & Chorilli M. (2022). Fabrication of Functional bioMOF-100 Prototype as Drug Delivery System for Breast Cancer Therapy. Pharmaceutics, 14(11), 2458.

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Annexin V and PI Apoptosis Assay

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CLL lymphocyte cells were left untreated or treated with DMSO alone; 0.5 nM ABT-199 or 1 nM ABT-737; 3 or 10 μM AZD1208, SMI-4a, or SGI-1776; or a combination of a Bcl-2 antagonist and a Pim kinase inhibitor at the indicated concentrations. Cells were washed, resuspended in 200 μL of Annexin binding buffer (Roche, Indianapolis, IN), mixed with 5 μL of Annexin V solution (BD Pharmingen, San Diego, CA) plus 5 μL of propidium iodide (PI; Sigma-Aldrich, St. Louis, MO), and incubated for 15 min in the dark at room temperature. At least 1 x 10⁴ cells were measured per sample using a Becton Dickinson FACSCalibur flow cytometer (San Jose, CA).

Cervantes-Gomez F., Lavergne B., Keating M.J., Wierda W.G, & Gandhi V. (2015). Combination of Pim kinase inhibitors and Bcl-2 antagonists in chronic lymphocytic leukemia cells. Leukemia & lymphoma, 57(2), 436-444.

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Annexin V/PI Apoptosis Assay

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Cells were untreated or treated with DMSO or AZD1208 and cell death (Annexin V/propidium iodide (PI) positivity) was measured using a Becton Dickinson FACSCalibur flow cytometer (Franklin Lakes, NJ, USA). Briefly, cells were washed, suspended in Annexin binding buffer (200 μL) (Roche; Indianapolis, IN, USA), mixed with Annexin V solution (5 μL) (BD Pharmingen; San Diego, CA, USA) plus PI (5 μL) (Sigma-Aldrich; St. Louis, MO, USA) and incubated for 15 min in the dark at room temperature. At least 10,000 cells were measured per sample.

Cervantes-Gomez F., Stellrecht C.M., Ayres M.L., Keating M.J., Wierda W.G, & Gandhi V. (2019). PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells. Oncotarget, 10(29), 2793-2809.

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Quantifying Hepatocyte Apoptosis

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Apoptosis was detected through the terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay or via PI/annexin V staining. For the TUNEL assay, frozen liver sections were permeabilized and incubated with the TUNEL test solution (Beyotime) for 60 min. Fluorescence was captured using a laser confocal microscope. For PI/annexin V staining, cells were resuspended in 100 μl of binding buffer containing 5 μl of PI and 10 μl of annexin V solution (BD Biosciences, USA) and incubated at 25℃ for 15 min. The number of apoptotic cells was measured via flow cytometry (ACEA NovoCyte, USA).

Yang Y., Fan S., Chen Q., Lu Y., Zhu Y., Chen X., Xia L., Huang Q., Zheng J, & Liu X. (2022). Acute exposure to gold nanoparticles aggravates lipopolysaccharide-induced liver injury by amplifying apoptosis via ROS-mediated macrophage-hepatocyte crosstalk. Journal of Nanobiotechnology, 20, 37.

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Apoptosis Assessment of Dissociated Spheroids

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After spheroid dissociation, single-cell suspensions were treated with Annexin V Binding Buffer 1X (BD Biosciences, San Jose, California, USA), and stained with 5- Fluorescein Isothiocyanate (FITC) conjugated with Annexin V solution (BD Biosciences, San Jose, California, USA) and Propidium Iodide solution (PI) (BD Biosciences, San Jose, California, USA) in order to estimate the percentage of apoptotic cells by using the FACSCalibur flow cytometer (BD Biosciences, San Jose, California, USA). Samples were analyzed in quadruplicate.

Amaral R.L., Miranda M., Marcato P.D, & Swiech K. (2017). Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening. Frontiers in Physiology, 8, 605.

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Autophagy and Apoptosis Quantification

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Cell autophagy was assessed by detecting the mono-dansylcadaverine (MDC; Sigma-Aldrich; Merck KGaA) positively stained cells using flow cytometry. Cells (10⁴) were washed with PBS and then incubated with 0.05 mmol/l MDC in PBS at 37°C for 45 min. After washing three times with PBS, the cell suspension was analyzed by flow cytometry immediately. For cell apoptosis detection, 10⁴ cells were collected in 200 ml RPMI-1640 medium without FBS. Following resuspension, approximately 10 ml Annexin V solution (BD Biosciences) was added. Fifteen min later, 300 ml medium buffer and 5 ml propidium iodide (PI; BD Biosciences) were added, and the cell suspension was analyzed by flow cytometry (BD Biosciences) immediately (11 (link)).

Qiao P.F., Yao L, & Zeng Z.L. (2020). Catalpol-mediated microRNA-34a suppresses autophagy and malignancy by regulating SIRT1 in colorectal cancer. Oncology Reports, 43(4), 1053-1066.

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