Annexin 5 phycoerythrin pe

Manufactured by BD

Sourced in United States, Belgium

Annexin V-Phycoerythrin (PE) is a fluorescent conjugate used for the detection and quantification of apoptotic cells. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is exposed on the surface of cells undergoing apoptosis. The Phycoerythrin (PE) fluorescent dye is used to label the Annexin V, allowing for the visualization and analysis of apoptotic cells using flow cytometry or other fluorescence-based techniques.

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12 protocols using annexin 5 phycoerythrin pe

Cell Cycle and Apoptosis Analysis of Gimatecan and Irinotecan

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To analyze the changes in cell cycle progression, cell pellets were harvested after exposure to gimatecan or irinotecan for 0 h, 4 h, 8 h, and then fixed in 70% cold ethanol at 4 °C overnight. Then the fixed cells were stained with 50 μg/mL propidium iodide (BD Biosciences), and incubated at room temperature in darkness for 30 min. Flow cytometry was performed using a FACS Calibur system (BD Biosciences) and data were analyzed by ModFit 4.0 software (BD Biosciences).
For cell apoptosis analysis, cells were exposed to gimatecan or irinotecan for 24 h, 48 h, or 72 h, and then stained with Annexin V- phycoerythrin PE and 7-amino-actinomycin (7-AAD) (BD Biosciences, Erembodegem, Belgium) at room temperature in the dark for 15 min. The flow cytometric analyses were conducted within 30 min. Cell apoptotic data were analyzed using the FlowJo software (TreeStar, Inc., Ashland, OR).

Zou J., Li S., Chen Z., Lu Z., Gao J., Zou J., Lin X., Li Y., Zhang C, & Shen L. (2018). A novel oral camptothecin analog, gimatecan, exhibits superior antitumor efficacy than irinotecan toward esophageal squamous cell carcinoma in vitro and in vivo. Cell Death & Disease, 9(6), 661.

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Survival Assay for Stimulated DNMT3A KO CAR T Cells

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Survival of serially stimulated DNMT3A KO CAR T cells in the absence of tumor was assessed by the positive selection of CD3⁺ coculture cells (Miltenyi Biotec) followed by a 7-day culture in the absence of tumor cells with or without IL-15. Live and dead cells were enumerated by flow cytometry. Samples were washed with PBS, stained with a 1:1000 dilution of viability dye LDA (Invitrogen) or eFluor 520 (Invitrogen), washed with PBS, and resuspended in 1× annexin V staining buffer (BD Biosciences) with 5 μl of annexin V phycoerythrin (PE) or annexin V allophycocyanin (APC) (BD Biosciences). Data were acquired on a BD FACSCanto and analyzed using FlowJo version 10.5.3.

Prinzing B., Zebley C.C., Petersen C.T., Fan Y., Anido A.A., Yi Z., Nguyen P., Houke H., Bell M., Haydar D., Brown C., Boi S.K., Alli S., Crawford J.C., Riberdy J.M., Park J.J., Zhou S., Velasquez M.P., DeRenzo C., Lazzarotto C.R., Tsai S.Q., Vogel P., Pruett-Miller S.M., Langfitt D.M., Gottschalk S., Youngblood B, & Krenciute G. (2021). Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity. Science translational medicine, 13(620), eabh0272.

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Apoptosis Analysis in Cell Cultures

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Dulbecco’s modified Eagle’s medium (DMEM), low-melting-point agarose (LMP), high-melting-point agarose (HMP), phosphate-buffered saline (PBS; Na₂HPO₄, KH₂PO₄ and KCl, pH 7.4), propidium iodide (PI), mitoxantrone (MXT), hydrogen peroxide (H₂O₂), amino acids and nitrogenated bases were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). Primary antibody anti-caspase-9 and secondary antibody anti-rabbit IgG (H + L) F(ab’)2 fragment conjugated to Alexa Fluor® 488 were obtained from Cell Signaling Technology (USA) and Invitrogen (Grand Island, NY, USA), respectively. Cell Proliferation Kit II (XTT) was acquired from Roche (Basel, Switzerland). Annexin V-Phycoerythrin (PE) and 7-Amino-Actinomycin (7-AAD) were purchased from BD Biosciences (San Diego, CA). Yeast extract, bacto-peptone, bacto-agar and yeast nitrogen base were obtained from Difco Laboratories (Detroit, MI). All other reagents were of analytical grade.

Viau C.M., Moura D.J., Facundo V.A, & Saffi J. (2014). The natural triterpene 3β,6β,16β-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells. BMC Complementary and Alternative Medicine, 14, 280.

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ER Stress and Apoptosis Regulation

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CD4 (L3T4) microbeads were provided by Miltenyi Biotec Company, Bergisch Gladbach, Germany. Lipopolysaccharide [Escherichia coli O127:B8, lipopolysaccharide (LPS)] was procured from Sigma-Aldrich, St Louis, MO, USA. Concanavalin A (Con A) was provided by Solarbio, Beijing, China. Annexin-V-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD) apoptosis detection kits were purchased from BD, San Diego, CA, USA. An anti-NUFIP1 antibody was obtained from Proteintech, Rosemont, IL, USA. Antibodies specific for protein kinase RNA-like ER kinase (PERK), phosphorylated (p)-PERK, C/EBP homologous protein (CHOP), Bcl-2, Bax, and cleaved (c)-Caspase3 were obtained from Cell Signaling Technology, Danvers, MA, USA. Antibodies specific for glucose-regulated protein 78 (GRP78), ribosomal protein L7 (RPL7) and RPL26 were obtained from Abcam, Cambridge, MA, USA. ER-Tracker red was obtained from Invitrogen, Carlsbad, CA, USA and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) and Alexa Fluor 594-conjugated goat anti-mouse IgG (H + L) were provided by Santa Cruz Biotechnology, Santa Cruz, CA, USA. Triton X-100 was purchased from Sigma, St. Louis, MO, USA and salubrinal (Sal) was obtained from Selleckchem, Houston, TX, USA. Hoechst 33258 was procured from APExBIO, Houston, TX, USA.

Zhao P.Y., Yao R.Q., Zheng L.Y., Wu Y., Li Y.X., Dong N., Li J.Y., Du X.H, & Yao Y.M. (2023). Nuclear fragile X mental retardation-interacting protein 1-mediated ribophagy protects T lymphocytes against apoptosis in sepsis. Burns & Trauma, 11, tkac055.

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Annexin V-PE and 7-AAD Apoptosis Assay

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Apoptosis was assessed using the annexin V-phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) apoptosis detection kit (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. After treatment with 10 µM cisplatin for 24 hours, SiHa cells were collected, washed with phosphate buffer saline twice and digested by 0.25% trypsin and dissociated into single cell. Then the cells were double-stained with 5 µL annexin V-PE and 7-AAD. Stained cells were analyzed by flow cytometry (BD). The experiment was repeated three times.

Zhao C., Lu E., Hu X., Cheng H., Zhang J.A, & Zhu X. (2018). S100A9 regulates cisplatin chemosensitivity of squamous cervical cancer cells and related mechanism. Cancer Management and Research, 10, 3753-3764.

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Apoptosis Analysis of AZD1775 and Cisplatin

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Cells exposed to AZD1775 with/without cisplatin for 48 h were collected, washed in phosphate-buffered saline (PBS), and double-stained using Annexin V-Phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) apoptosis detection kit (BD Biosciences, Erembodegem, Belgium) following the vendor's protocol. Samples were detected by flow cytometry within 1 h (BD Biosciences) and proportions of apoptotic cells were analyzed using the FlowJo version 7.6.1 software (FlowJo, Oregon).

Chen D., Lin X., Gao J., Shen L., Li Z., Dong B., Zhang C, & Zhang X. (2018). Wee1 Inhibitor AZD1775 Combined with Cisplatin Potentiates Anticancer Activity against Gastric Cancer by Increasing DNA Damage and Cell Apoptosis. BioMed Research International, 2018, 5813292.

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Apoptosis Quantification by Flow Cytometry

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After drug treatment, cells were double-stained with Annexin V-phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) (BD Biosciences) at room temperature in the dark as described in the vendor’s protocol, followed by flow cytometry analysis within 1 h (BD Biosciences). We analyzed proportions of apoptotic cells using FlowJo Version 7.6.1 software (FlowJo, Ashland, OR).

Lin X., Peng Z., Wang X., Zou J., Chen D., Chen Z., Li Z., Dong B., Gao J, & Shen L. (2019). Targeting autophagy potentiates antitumor activity of Met-TKIs against Met-amplified gastric cancer. Cell Death & Disease, 10(2), 139.

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Quantifying CD71+ and Phosphatidylserine-Positive RBCs

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To measure the amount of CD71+ and of phosphatidylserine positive (P+) RBC, arterial blood was collected in heparinized tubes and processed within 2 h. 30 µL blood was diluted with 15 mL ice-cold phosphate buffered saline (PBS). RBC suspensions were stained with 5 µL CD71-Allophycocyanin (APC) (#130-091-727, Milteny Biotech) and Annexin-V-Phycoerythrin (PE) (#556422, BD Bioscience) for 30 min at 4 °C in the dark. All tubes were centrifuged (300g, 10 min, 4 °C), and supernatants were removed. Pellets were resuspended in 1000 µL PBS and analysed via flow cytometry (BD FACSVerse™ flow cytometer, BD Biosciences, San Jose, California, USA). Flow cytometric data were collected using the BD FACSuite, analysed using FlowJo (TreeStar), and calculated as described above. The number of CD71+ or PS+ was calculated as percentage of total gated RBC. Median fluorescence intensity (MFI) was calculated from the histogram (distribution) plots of the green fluorescence signals [extinction (Ex) 488 nm, emission (Em) 530 ± 30 nm] detected within the cell-specific gates. MFIs of untreated samples served as autofluorescence controls [10 (link)].

Wischmann P., Kuhn V., Suvorava T., Muessig J.M., Fischer J.W., Isakson B.E., Haberkorn S.M., Flögel U., Schrader J., Jung C., Cortese-Krott M.M., Heusch G, & Kelm M. (2020). Anaemia is associated with severe RBC dysfunction and a reduced circulating NO pool: vascular and cardiac eNOS are crucial for the adaptation to anaemia. Basic Research in Cardiology, 115(4), 43.

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Apoptosis Quantification by Flow Cytometry

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After drug treatment for 48 h, cells were double-stained with Annexin V-Phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) (BD Biosciences) at room temperature in the dark as described in the vendor’s protocol, followed by flow cytometry analysis within 1 h (BD Biosciences). We analyzed proportions of apoptotic cells using FlowJo Version 7.6.1 software (FlowJo, Ashland, OR).

Chen D., Lin X., Zhang C., Liu Z., Chen Z., Li Z., Wang J., Li B., Hu Y., Dong B., Shen L., Ji J., Gao J, & Zhang X. (2018). Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway. Cell Death & Disease, 9(2), 123.

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Apoptosis Assessment of HNSC Cells After PFN2 Transduction

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Apoptosis of HNSC cells after PFN2 transduction was assessed by Annexin-V–phycoerythrin (PE) and 7-amino-actinomycin D (7-AAD) apoptosis detection kit (BD, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. Briefly, cells were treated with 10 μM cisplatin for 24 h, and then the cells were collected and washed twice with cold PBS. After that, the cells were stained with 5 μl of annexin V and 5 μl of 7-AAD for 15 min in the dark. Cell apoptosis was analyzed by CytoFLEX flow cytometer.

Zhou K., Chen J., Wu J., Xu Y., Wu Q., Yue J., Song Y., Li S., Zhou P., Tu W., Yang G, & Jiang S. (2019). Profilin 2 Promotes Proliferation and Metastasis of Head and Neck Cancer Cells by Regulating PI3K/AKT/β-Catenin Signaling Pathway. Oncology Research, 27(9), 1079-1088.

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