Annexin 5 conjugated alexa fluor 488 apoptosis detection kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Annexin V-conjugated Alexa Fluor 488 apoptosis detection kit is a laboratory reagent designed to detect and quantify apoptosis. It utilizes Annexin V, a protein that binds to phosphatidylserine, a molecule exposed on the surface of cells undergoing apoptosis. The Annexin V is conjugated to the fluorescent dye Alexa Fluor 488, allowing for the visualization and analysis of apoptotic cells using flow cytometry or fluorescence microscopy.

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5 protocols using annexin 5 conjugated alexa fluor 488 apoptosis detection kit

Proanthocyanidins Modulate β-Catenin Signaling

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The purified fraction of proanthocyanidins from grape seeds were obtained from the Kikkoman Corp. (Noda, Japan). The β-catenin^S33Y pcDNA plasmid bearing FLAG tag used for the overexpression of non-degradable, constitutively active mutant form of β-catenin was obtained from Addgene (Cambridge, MA, USA), while β-catenin siRNA kit for knocking down the expression level of β-catenin along with the siRNA transfection reagents, and antibodies specific to PCNA, cyclin D1, cyclin D2, Cdks (2 (link),4 (link),6 (link)), Cip1/p21, Kip1/p27, β-catenin, β-actin, histone H3, horseradish peroxidase conjugated rabbit anti-goat and goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for Bax, Bcl-2, Bcl-xl, cleaved caspase-3, caspase-9, PARP, casein kinase 1α (CK1α), glycogen synthase kinase-3β (GSK-3β), and phospho forms of β-catenin were obtained from Cell Signaling Technology (Beverly, MA, USA). Annexin V-conjugated Alexa Fluor 488 apoptosis detection kit was purchased from Molecular Probes, Inc. (Eugene, OR, USA).

VAID M., SINGH T., PRASAD R, & KATIYAR S.K. (2015). Bioactive proanthocyanidins inhibit growth and induce apoptosis in human melanoma cells by decreasing the accumulation of β-catenin. International Journal of Oncology, 48(2), 624-634.

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Honokiol Inhibits EGFR-Mediated Apoptosis Signaling

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Purified honokiol was purchased from Quality Phytochemicals LLC (Edison, NJ). Antibodies specific for Bax, Bcl-2, Bcl-xl, cleaved caspase 3, cyclin D1, cyclin D2, mTOR, 4E-BP1, p70S6 kinase, pEGFR and EGFR were obtained from Cell Signaling Technology (Beverly, MA). Gefitinib and antibodies specific for PCNA, cyclin-dependent kinases (Cdk), β-actin and horseradish peroxidase conjugated rabbit anti-goat and goat anti-rabbit secondary antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). The Annexin V-conjugated AlexaFluor488 Apoptosis Detection Kit was purchased from Molecular Probes, Inc. (Eugene, OR). The protein assay kit was procured from Bio-Rad (Hercules, CA). 3-[4,5-dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), rapamycin and all other chemicals were of analytical grade and obtained from Sigma Chemical Co. (St. Louis, MO).

Singh T., Gupta N.A., Xu S., Prasad R., Velu S.E, & Katiyar S.K. (2015). Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor. Oncotarget, 6(25), 21268-21282.

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Quantification of Apoptotic and Necrotic Cells

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Apoptotic and necrotic cells were quantified using the flow cytometric analysis and an Annexin V-conjugated Alexa Fluor 488 Apoptosis Detection Kit (Molecular Probes, Eugene, OR, USA) based on the manufacturer’s instructions. To detect the percentage of apoptotic cells, the samples were stained simultaneously with Alexa Fluor^® 488 dye and propidium iodide (PI). The percentage of apoptotic cells were quantified using flow cytometry (Coulter Epics XL-MCL; Beckman Coulter, Indianapolis, IN, USA). These cell populations can be distinguished using a flow cytometer with the 488 nm line of an argon-ion laser for excitation.

Sulistyowati E., Hsu J.H., Lee S.J., Huang S.E., Sihotang W.Y., Wu B.N., Dai Z.K., Lin M.C, & Yeh J.L. (2022). Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo. International Journal of Molecular Sciences, 23(10), 5673.

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Annexin V-Alexa Fluor 488 Apoptosis Assay

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Briefly, cells were collected in phosphate-buffered saline (PBS) and resuspended in annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4). Cells were stained with 5 μL Annexin V-Alexa Fluor 488 and propidium iodide (PI, 1 μg/mL) at room temperature for 15 min in the dark. Apoptotic cell death was analyzed by flow cytometry using the Annexin V-conjugated Alexa Fluor488 Apoptosis Detection Kit according the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA). Stained cells were analyzed by FACScan flow cytometry using CellQuest 3.3 analysis software (Becton Dickinson, San Jose, CA, USA) [33 (link)].

Liu J.J., Wu H.H., Chen T.H., Leung W, & Liang Y.C. (2015). 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies. International Journal of Molecular Sciences, 16(8), 19387-19400.

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Apoptosis and Protein Expression Assays

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Trichostatin A (TSA) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). The protein assay kit was from Bio-Rad (Hercules, CA, USA). Annexin-V conjugated AlexaFluor488 Apoptosis Detection Kit from Molecular Probes, Inc., (Eugene, OR). p16 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MUC4 monoclonal antibody (8G7) was developed in our laboratory.^{53 (link)} Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG were procured from GE Healthcare Biosciences (Uppsala, Sweden) and FITC-conjugated anti-mouse IgG was obtained from Invitrogen (California, USA). All other antibodies used are listed in supplementary table 1.

Macha M.A., Rachagani S., Pai P., Gupta S., Lydiatt W.M., Smith R.B., Johansson S.L., Lele S.M., Kakar S.S., Lee J.H., Meza J., Ganti A.K., Jain M, & Batra S.K. (2014). MUC4 Regulates Cellular Senescence in Head and Neck Squamous Cell Carcinoma (HNSCC) through p16/Rb Pathway. Oncogene, 34(13), 1698-1708.

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