Annexin 5 phycoerythrin pe apoptosis detection kit

Manufactured by BD

Sourced in United States

The Annexin V-phycoerythrin (PE) apoptosis detection kit is a laboratory instrument used to detect and quantify apoptosis, a type of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule expressed on the surface of apoptotic cells. The Annexin V is conjugated to the fluorescent dye phycoerythrin (PE), allowing for the detection and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.

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PE Annexin V Apoptosis Detection Annexin V Apoptosis Detection Kit Annexin V-Phycoerythrin (PE) Annexin V-phycoerythrin (Annexin V-PE; Annexin V Apoptosis Kit Annexin V-Phycoerythrin Annexin V detection kit Annexin V-PE Apoptosis detection kit Annexin V kit

14 protocols using annexin 5 phycoerythrin pe apoptosis detection kit

Cell Cycle and Apoptosis Analysis

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For the cell cycle assay, cells were gathered at 48 h, washed in ice-cold PBS and fixed in ice-cold 70% ethanol overnight. Following removal of the ethanol, the cells were stained in the dark with 50 µg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA) and 100 µg/ml RNase for 30 min at room temperature. The stained cells were examined for DNA content by Epics XL flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). The result was analyzed by ModFit LT software (version 3.1, Verity Software House Inc., Topsham, ME, USA).
To identify apoptosis, flow cytometry analysis was performed with an Annexin V-Phycoerythrin (PE) Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA). Cells were stained with 5 µl Annexin V-PE, 5 µl 7-aminoactinomycin D (AAD), and 500 µl 1X binding buffer for 15 min, and subsequently examined by Epics XL flow cytometry. The result was analyzed by CXP software (version 2.1, Beckman Coulter, Inc., Brea, CA, USA).

Xu L., Tang H., Wang K., Zheng Y., Feng J., Dong H., Jin Y., Cao C., Chen X, & Gao G. (2019). Pharmacological inhibition of EZH2 combined with DNA-damaging agents interferes with the DNA damage response in MM cells. Molecular Medicine Reports, 19(5), 4249-4255.

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Annexin V-PE Apoptosis Assay

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Cells (1 × 10⁶) were seeded in 100 mm plates and allowed to attach overnight. Next cells were treated with 1 μM (IC25) and 5 μM (IC50) PC for 6 h. Following treatment cells were harvested and stained using Annexin V-phycoerythrin (PE) apoptosis detection kit (BD Pharmingen) according to the manufacturer’s recommendations. Briefly, the cells were trypsinized, washed in PBS, and resuspended in binding buffer (1 × 10⁶/mL). Equal volumes of Annexin V-PE and 7-amino-actinomycin D (7-AAD) were added and incubated for 15 min at room temperature in the dark and analyzed by flow-cytometry.

Ravi M., Tentu S., Baskar G., Rohan Prasad S., Raghavan S., Jayaprakash P., Jeyakanthan J., Rayala S.K, & Venkatraman G. (2015). Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells. BMC Cancer, 15, 768.

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Autophagy and Apoptosis Evaluation

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Cell lines were maintained in DMEM medium supplemented with 10% FCS were sourced from Corning Cellgro Inc (Herndon, VA, USA). A Cyto-ID autophagy detection kit was sourced from Enzo Life Sciences Inc (Farmingdale, NY, USA). 4, 6-Diamidino-2-phenylindole (DAPI) was obtained from Invitrogen (Carlsbad, CA, USA). The thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma-Aldrich Inc (St Louis, MO, USA). An Annexin V: phycoerythrin (PE) apoptosis detection kit was obtained from BD Biosciences Inc (San Jose, CA, USA). Primary antibodies against human were all purchased from Cell Signaling Technology Inc (Beverly, MA, USA). The antibody against human β-actin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).

Yao M., Shang Y.Y., Zhou Z.W., Yang Y.X., Wu Y.S., Guan L.F., Wang X.Y., Zhou S.F, & Wei X. (2017). The research on lapatinib in autophagy, cell cycle arrest and epithelial to mesenchymal transition via Wnt/ErK/PI3K-AKT signaling pathway in human cutaneous squamous cell carcinoma. Journal of Cancer, 8(2), 220-226.

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Cisplatin-Induced Apoptosis and Autophagy

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CDDP was purchased from Selleckchem Inc. (Houston, TX, USA). ¹³C₆-L-lysine, L-lysine, ¹³C₆¹⁵N₄-L-arginine, L-arginine, Dulbecco’s modified Eagle’s medium (DMEM)/F12 for SILAC, APE1 siRNA, dimethyl sulfoxide (DMSO), 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, and Dulbecco’s phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 6-Diamidino-2-phenylindole (DAPI), Opti-minimal Essential Medium (MEM), Lipofectamine 2000, and the negative control siRNA were purchased from Invitrogen Inc. (Carlsbad, CA, USA). The Annexin V-phycoerythrin (PE) apoptosis detection kit was purchased from BD Biosciences Inc. (San Jose, CA, USA). The Cyto-ID® Autophagy detection kit was obtained from Enzo Life Sciences Inc. (Farmingdale, NY, USA). The Western blotting substrate, Pierce™ bicinchoninic acid (BCA) protein assay kit, skim milk, and radioimmunoprecipitation assay buffer (RIPA) were purchased from Thermo Fisher Scientific Inc. (Hudson, NH, USA). The polyvinylidene difluoride (PVDF) membrane was obtained from Bio-Rad Inc. (Hercules, CA, USA). The antibody against human β-actin was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The remaining primary antibodies for signalling proteins related to apoptosis and autophagy were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Pan S.T., Zhou J., Yang F., Zhou S.F, & Ren T. (2020). Proteomics reveals a therapeutic vulnerability via the combined blockade of APE1 and autophagy in lung cancer A549 cells. BMC Cancer, 20, 634.

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Apoptosis Detection by Flow Cytometry

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Cells were harvested 48 hrs after transfection. The percentage of apoptotic cells was determined using Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) under FACS Calibur (BD Biosciences) according to the manual. The data were assessed with Flowjo software (Tree Star Corp, San Carlos, CA, USA).

Cui X., Yu T., Shang J., Xiao D, & Wang X. (2019). Long Non-Coding RNA CDKN2B-AS1 Facilitates Laryngeal Squamous Cell Cancer Through Regulating miR-497/CDK6 Pathway. OncoTargets and therapy, 12, 8853-8862.

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Annexin V-PE Apoptosis Assay Protocol

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Apoptotic cell death rate was assessed by using the annexin V-phycoerythrin (PE) apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, the cells were harvested by trypsinization, centrifuged, and washed twice in cold PBS. Afterwards, they were resuspended in 100 µL of 1× binding buffer at a concentration of 1 × 10⁶ cells/mL. After the addition of 5 µL of annexin V PE and 5 µL of 7-amino-actinomycin (7-AAD), the cells were gently vortexed and incubated for 15 min at room temperature (25°C) in dark. The cell samples were diluted in 400 µL of 1 × binding buffer and analyzed by flow cytometry within 1 h using a Nucleocounter NC-3000 instrument (ChemoMetec Inc, Allerod, Denmark). Compensation and plot quadrants were determined by using the unstained cells and cells stained with either annexin V-PE or 7-AAD alone. The gating strategy was as follows: an initial forward scatter vs. side scatter gating, to exclude debris, followed by annexin V-PE and 7-AAD gating, to distinguish the apoptotic from viable cells. By using this gating strategy, annexin V-negative/7-ADD negative cells were defined as viable, annexin-positive/7-AAD negative cells as early apoptotic, annexin V-negative/7-ADD-positive cells as dead cells, and annexin V-positive/7-ADD-positive cells as late apoptotic or necrotic cells.

Chin L.H., Hsu Y.J., Hsu S.C., Chen Y.H., Chang Y.L., Huang S.M., Tsai C.S, & Lin C.Y. (2017). The regulation of NLRP3 inflammasome expression during the development of cardiac contractile dysfunction in chronic kidney disease. Oncotarget, 8(69), 113303-113317.

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Apoptosis detection in oridonin-treated cells

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The percentage of apoptotic of cells exposed to oridonin (25 µM) with or without SS31 (100 nM) for 8 h were determined using a commercially available Annexin V-phycoerythrin (PE) apoptosis detection kit according to the manufacturer's protocol (BD Biosciences, Franklin Lakes, NJ, USA) with minor modifications. Briefly, cells were collected and washed twice in ice cold PBS and then resuspended in 100 µl 1X binding buffer at a density of 1×10⁵ cells/ml, and then incubated with 5 µl Annexin V-PE and 5 µl 7-aminoactinomycin (7-AAD) in the dark for 15 min at room temperature. Finally, 400 µl of 1X binding buffer was added to each tube. Samples were analyzed using a BD FACSCalibur (BD Biosciences) and BD CellQuest Pro software (version 5.1; BD Biosciences) and evaluated based on the percentage of apoptotic cells that were Annexin V-PE-positive and 7-AAD-positive.

Liu X., Kang J., Wang H, & Huang T. (2017). Mitochondrial ROS contribute to oridonin-induced HepG2 apoptosis through PARP activation. Oncology Letters, 15(3), 2881-2888.

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Annexin V-PE Apoptosis Assay by Flow Cytometry

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Apoptotic cells were analyzed by using an Annexin V-Phycoerythrin (PE) Apoptosis Detection kit (BD Biosciences) and flow cytometric analysis (FACSAria II; BD Biosciences). Untransfected Eca109 cells or Eca109 cells transfected with siRNA or control-siRNA for 48 h were re-suspended in 1X working solution at 1×10⁶ cells/ml. Following addition of 5 µl Annexin V-PE and 5 µl 7-amino-actinomycin stain (7-AAD; eBioscience, San Diego, CA, USA), cells were incubated at 25°C in the dark for 15 min. Subsequently, 400 µl binding buffer was added and cells were analyzed by flow cytometry within 1 h of staining.

QIAO L., LIANG N., XIE J., LUO H., ZHANG J., DENG G., LI Y, & ZHANG J. (2015). Gene silencing of galectin-3 changes the biological behavior of Eca109 human esophageal cancer cells. Molecular Medicine Reports, 13(1), 160-166.

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Quantification of Apoptosis by Annexin V-PE

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Apoptosis was measured with Annexin V-phycoerythrin (PE) Apoptosis Detection kit (BD Biosciences). The cells (2×10⁵ cells/well) were plated in 6-well plates for 24 h. Then, the cells were washed with cold PBS and resuspended in 100 µl 1X binding buffer, followed by the addition of 5 µl Annexin V-PE and 5 µl 7-aminoactinomycin D (7-AAD). The cells were incubated for 15 min at room temperature in the dark. Finally, 380 µl 1X binding buffer was added to the cells. Cell apoptosis was analyzed using a flow cytometer and CXP analysis software 2.2 (Beckman Coulter, Inc., Brea, CA, USA).

Wu H., Li X., Feng M., Yao L., Deng Z., Zao G., Zhou Y., Chen S, & Du Z. (2018). Downregulation of RNF138 inhibits cellular proliferation, migration, invasion and EMT in glioma cells via suppression of the Erk signaling pathway. Oncology Reports, 40(6), 3285-3296.

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Apoptosis Analysis of Transfected Cells

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After transfection, the percentage of apoptotic cells was analyzed by using Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (BD Biosciences, NY, USA) according to the manual. In brief, the transfected HepG2 cells with sh-ANRIL#1, sh-ANRIL#2, and miR-191 mimic were harvested after transfection for 48 h. These cells were then washed twice with phosphate buffered saline (PBS, Gibco). After this, 5 μl Annexin V-PE was added to stain these cells for 15 min in the dark at room temperature. Cell apoptosis was finally analyzed by using flow cytometry analysis (FACSC, alibur, BD, USA).

Huang D., Bi C., Zhao Q., Ding X., Bian C., Wang H., Wang T, & Liu H. (2018). Knockdown long non-coding RNA ANRIL inhibits proliferation, migration and invasion of HepG2 cells by down-regulation of miR-191. BMC Cancer, 18, 919.

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