Annexin 5 pe kit

Manufactured by BD

Sourced in United States

The Annexin V-PE kit is a laboratory reagent used for the detection and quantification of apoptosis, a programmed cell death process. The kit contains Annexin V conjugated with the fluorescent dye phycoerythrin (PE), which binds to phosphatidylserine, a molecule exposed on the surface of apoptotic cells. This allows for the identification and analysis of apoptotic cells by flow cytometry or other fluorescence-based techniques.

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Annexin V (870 citations) Annexin V-PE (283 citations) Annexin V kit (54 citations) Annexin V PE/7AAD kits (2 citations) FITC/PE Annexin V Apoptosis Detection Kits (2 citations)

17 protocols using annexin 5 pe kit

Cell Proliferation and Apoptosis Assays

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Cell proliferation was determined by cell counting with a Neubauer hemocytometer as described in the figure legends. Alternatively, viable cell amounts were measured using Cell Titer-Blue (Promega, Walldorf, Germany). To this end, 5 × 10³ or 3 × 10⁴ (for murine cell lines or human AML cell lines, respectively) cells were seeded in 100 µL growth medium per well into 96-well black plates (Greiner, Frickenhausen, Germany), and complemented with 25 µL growth medium with different concentrations of drugs. After incubation for 3 days, 25 µL Cell Titer-Blue reagent was added into each well and plates were incubated for another 3 h. The fluorescence signal was measured with a TECAN Infinite 200 plate reader at excitation and emission wavelengths of 540 nm and 610 nm, respectively.
Apoptosis assays were performed using the Annexin-V method. Cells were treated with the drug or drug combinations for 48 h and detection of apoptotic cells was carried out with a PE annexin-V kit (AB_2869265, BD Biosciences, Heidelberg, Germany) according to the instructions of the manufacturer.

Demircan M.B., Mgbecheta P.C., Kresinsky A., Schnoeder T.M., Schröder K., Heidel F.H, & Böhmer F.D. (2022). Combined Activity of the Redox-Modulating Compound Setanaxib (GKT137831) with Cytotoxic Agents in the Killing of Acute Myeloid Leukemia Cells. Antioxidants, 11(3), 513.

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Apoptosis Assessment of SAHA and 5c Compounds

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HEL (1 × 10⁵/well) cells were incubated in six-well plates for 24 h and then treated with 0.1% DMSO (as control), various doses of SAHA, or 5c (0.5, 1, 2 µM) in fresh growth medium. After 24 h, the growth medium was collected, and the cells were trypsinized and collected with the corresponding medium. After centrifugation at 2000 rpm at 4 °C for 5 min, the supernatant was removed completely, and the cells were washed twice with prechilled PBS. A 200 µL volume of 1× binding buffer, 2.5 µL of 7-AAD, and 2.5 µL of propidium iodide were added (PE-Annexin V kit, BD Pharmingen). The cells were gently vortex-mixed and incubated for 15 min at 25 °C in the dark. Using cells stained with 7-AAD and propidium iodide alone as the positive control, the samples were detected with a FACS Calibur flow cytometer (Becton Dickinson).

Duan W., Li J., Inks E.S., Chou C.J., Jia Y., Chu X., Li X., Xu W, & Zhang Y. (2015). Design, Synthesis, and Antitumor Evaluation of Novel Histone Deacetylase Inhibitors Equipped with a Phenylsulfonylfuroxan Module as a Nitric Oxide Donor. Journal of medicinal chemistry, 58(10), 4325-4338.

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Apoptosis and Oxidative Stress Evaluation

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The PE Annexin V Kit (BD, 559763, USA) was utilized to measure the apoptosis status of the cells. The tested cells were collected into centrifuge tubes and incubated with 7AAD and Annexin V dye for 15 min at 25°C. The prepared cells were identified by flow cytometry machine (Beckman, USA) with PE and PerCP fluorescence channel. Reactive Oxygen Species (ROS) detection kit (Beyotime, S0033S, China) was utilized to measure ROS in the cells. The BODIPY 581/591 C11 probe (Invitrogen, D3861, USA) was used to measure intracellular lipid peroxidation. The cells were incubated for 30 min after adding the probe. A flow cytometry machine with a FITC channel was used for evaluation. All results were analyzed using FlowJo software version 10.8.1.

Zhang H., Zhu S., Zhou H., Li R., Xia X, & Xiong H. (2023). Identification of MTHFD2 as a prognostic biomarker and ferroptosis regulator in triple-negative breast cancer. Frontiers in Oncology, 13, 1098357.

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Cell Proliferation and Apoptosis Assay

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Cell proliferation was assessed by BrdU incorporation assays with a BrdU Flow Kit (BD Pharmingen) or an EdU Click Proliferation Kit (BD Pharmingen). Cell apoptosis was analyzed with a PE-Annexin V Kit (BD Pharmingen). The following antibodies were purchased from BD Biosciences: CD122-PE (TM-Bta1), CD19-FITC (1D3), CD335/NKp46-PE (29A1.4), CD3e-FITC (145-2C11), CD3e-PerCP-Cy5.5 (145-2C11), CD4-FITC (RM4-5), CD49b (DX5) Pan-NK Cells-BV421, CD8a-PE (53-6.7), Erythroid Cells-FITC (TER-119), Ly-6G/Ly-6C-FITC (RB6-8C5), NK-1.1-APC (PK136), NK-1.1-APC-Cy7 (PK136), and TCR-Bta Chain-BV421 (H57-597). Stained samples were analyzed using FACSCantoII and FACSDiva software Version 6.1.2 (BD Biosciences).

Li Z., Zhang X., Xue W., Zhang Y., Li C., Song Y., Mei M., Lu L., Wang Y., Zhou Z., Jin M., Bian Y., Zhang L., Wang X., Li L., Li X., Fu X., Sun Z., Wu J., Nan F., Chang Y., Yan J., Yu H., Feng X., Wang G., Zhang D., Fu X., Zhang Y., Young K.H., Li W, & Zhang M. (2019). Recurrent GNAQ mutation encoding T96S in natural killer/T cell lymphoma. Nature Communications, 10, 4209.

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Cell Growth Inhibition Assay

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Cells were plated at equal concentrations with varying concentrations of the inhibitor and in triplicate wells. Cells were counted, the concentration was calculated every second/third day, and the cells were replated at even cell concentrations. A growth curve was plotted for cumulative growth according to the dilution at each time point. Doubling time was calculated from fitted semi-logarithmic slope (Nonlin). Fit using least squares in GraphPad Prism software by log₂/slope and Student’s two-tailed t test was used to test for significance. For cell cycle assays (Lp30 cells), cells were plated with 30 μM inhibitor or DMSO for 24 hours. Cells were harvested, and an equal number of cells were fixed in 2% paraformaldehyde, permeabilized in 0.1% saponin, stained with 4′,6-diamidino-2-phenylindole (DAPI), and analyzed by flow cytometry (LSR-II, BD Biosciences). For apoptosis assay (Lp30 cells), cells were plated with 30 μM inhibitor or DMSO for 72 hours, harvested, washed and stained using a PE-annexin V kit (BD Biosciences), and analyzed by flow cytometry (Accuri, BD Biosciences). All flow cytometry analyses were run with FlowJo software V.10.

Jakobsen J.S., Laursen L.G., Schuster M.B., Pundhir S., Schoof E., Ge Y., d’Altri T., Vitting-Seerup K., Rapin N., Gentil C., Jendholm J., Theilgaard-Mönch K., Reckzeh K., Bullinger L., Döhner K., Hokland P., Fitzgibbon J, & Porse B.T. (2019). Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML. Science Advances, 5(7), eaaw4304.

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Apoptosis Analysis of T Cells

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Flow cytometry analysis of T cell apoptosis was performed using the Annexin V-PE kit (BD Biosciences, San Jose, California, USA) as previously described (27 (link)). Briefly, T cells were incubated with various concentrations of rHcABHD proteins (0, 10, 20, 40, and 80 μg/mL) followed by staining with annexin V and 7-Aminoactinomycin D (7-AAD) based on the manufacturer's protocol. The PBS-treated goat T cells were served as negative controls. Three independent experiments were performed and each experiment was run in triplicate.

Lu M., Tian X., Tian A.L., Li C., Yan R., Xu L., Song X, & Li X. (2020). A Novel α/β Hydrolase Domain Protein Derived From Haemonchus contortus Acts at the Parasite-Host Interface. Frontiers in Immunology, 11, 1388.

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Apoptosis Assessment of Endothelial Cells

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Apoptosis level was measured using the dual-color Annexin V-PE kit (BD Biosciences Pharmigen, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. TECs and NECs were cultured in EGM-2 MV containing bLF (0.012, 0.12, 1.20 µM) for 3 days. The distribution of early apoptotic cells was analyzed from the dot plot quadrant marker using fluorescence-activated cell sorting (FACS Calibur Flow Cytometer; Becton-Dickinson, San Jose, CA, USA).

Ayuningtyas N.F., Chea C., Ando T., Saninggar K.E., Tanimoto K., Inubushi T., Maishi N., Hida K., Shindoh M., Miyauchi M, & Takata T. (2023). Bovine Lactoferrin Suppresses Tumor Angiogenesis through NF-κB Pathway Inhibition by Binding to TRAF6. Pharmaceutics, 15(1), 165.

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Cellular Senescence and Oxidative Stress

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Apoptosis and cellular senescence were assessed at third passage using Annexin-V-PE Kit (BD Biosciences) and β-Galactosidase (Cell Signaling Technology) respectively. DNA damage repair (DDR) potential was assessed by phosphorylation of γH2AX (Merck) and alkaline comet assay (Abcam). While reactive oxygen species (ROS) production was determined by measuring the formation of fluorescent dichloro fluorescein at an Excitation 485 and Emission 535 nm (Abcam).

Kutyna M.M., Kok C.H., Lim Y., Tran E.N., Campbell D., Paton S., Thompson-Peach C., Lim K., Cakouros D., Arthur A., Hughes T., Kumar S., Thomas D., Gronthos S, & Hiwase D.K. (2022). A senescence stress secretome is a hallmark of therapy-related myeloid neoplasm stromal tissue occurring soon after cytotoxic exposure. Leukemia, 36(11), 2678-2689.

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Annexin V-PE Assay for T Cell Apoptosis

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Flow cytometry assays were performed for T cell apoptosis determination using the Annexin V-PE kit (BD Biosciences, San Jose, CA, USA) as previously described [28 (link)]. In brief, freshly sorted T cells were cultured in the presence of tested doses of rHcADRM1 proteins (0, 5, 10, 20 and 40 µg/ml) followed by Annexin V and 7-aminoactinomycin D (7-AAD) staining based on the kit’s specification. The PBS-stimulated T cells served as negative controls. Three individual tests, each in triplicate, were conducted.

Lu M., Tian X., Zhang Y., Aimulajiang K., Wang W., Ehsan M., Li C., Yan R., Xu L., Song X, & Li X. (2020). Unveiling the immunomodulatory properties of Haemonchus contortus adhesion regulating molecule 1 interacting with goat T cells. Parasites & Vectors, 13, 424.

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Flow Cytometry Analysis of Murine Thymocytes

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Thymocytes from mice were surface stained in FACS Buffer (2% FBS in PBS) for 30 minutes on ice. Samples were washed with FACS Buffer and acquired on an LSRII flow cytometer (Becton Dickinson). Data were analyzed with FlowJo software (Becton Dickinson). Antibodies were from CD4 (clone GK1.5, BD Biosciences), CD8 (clone 53–6.7, eBiosciences), Tcr-β (clone H57–597, eBiosciences), CD71 (clone R17217, BioLegend), Live/dead (L34957, Molecular Probes), BrdU (8811–6600-42, eBiosciences). An Annexin-V PE kit (556422, BD Biosciences) was used to measure apoptosis according to the manufacturer’s instructions.

Emmanuel A.O., Arnovitz S., Haghi L., Mathur P.S., Mondal S., Quandt J., Okoreeh M.K., Maienschein-Cline M., Khazaie K., Dose M, & Gounari F. (2018). Tcf-1 and HEB cooperate to establish the epigenetic and transcription profile of CD4+CD8+ thymocytes. Nature immunology, 19(12), 1366-1378.

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