Annexin 5 alexa fluor 647 conjugate

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada

Annexin V Alexa Fluor 647 conjugate is a fluorescent dye-labeled protein that binds to phosphatidylserine, a phospholipid present on the surface of apoptotic cells. The Alexa Fluor 647 dye provides a bright, photostable fluorescent signal for detection of apoptosis.

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33 protocols using annexin 5 alexa fluor 647 conjugate

Cell Cycle & Apoptosis Analyses

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For cell cycle analysis, cells were harvested and fixed in 70% ethanol overnight at -20°C before staining with propidium iodide (PI) DNA staining solution (10 μg/ml PI, 100 μg/ml RNase A and 0.1% Triton-X 100 in PBS). For apoptosis analysis, cells were washed with Annexin V binding buffer (BD Pharmingen, NJ, USA) and stained with Annexin V Alexa Fluor 647 conjugate and SYTOX Blue at 1:1000 each (Thermo Scientific) before analysis using the LSR II Flow Cytometry System (BD Pharmingen).

Ho J.C., Abdullah L.N., Pang Q.Y., Jha S., Chow E.K., Yang H., Kato H., Poellinger L., Ueda J, & Lee K.L. (2017). Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway. PLoS ONE, 12(11), e0188051.

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Apoptosis Detection via Annexin V-Alexa Conjugate

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The phospholipid phosphatidyl serine (PS) was detected by the Annexin V–Alexa 633 conjugate after its externalization on the other side of the plasmatic membrane, which acts as a marker of programmed cell death. For apoptosis detection, Jurkat and HTL cells (1 × 10⁶) were harvested 3, 24, 48 or 72 h after PSE 45 µg/mL and Phy 60 µM treatment. NAC/PSE or NAC/Phy experimental groups were pre-treated with N-acetyl-L-cysteine (NAC c = 2 mM; Merck) for 1 h before PSE/Phy were applied. The cell suspension was washed in PBS and stained using Annexin V–Alexa Fluor^® 647 conjugate (Thermo Scientific) for 15 min at room temperature in the dark, followed by incubation with propidium iodide (PI, Merck) and analyses by flow cytometer (BD FACSCalibur). A minimum of 1 × 10⁴ events were analyzed per analysis, and all experiments were performed in triplicate.

Kello M., Kuruc T., Petrova K., Goga M., Michalova Z., Coma M., Rucova D, & Mojzis J. (2021). Pro-Apoptotic Potential of Pseudevernia furfuracea (L.) Zopf Extract and Isolated Physodic Acid in Acute Lymphoblastic Leukemia Model In Vitro. Pharmaceutics, 13(12), 2173.

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Plasmid-mediated gene delivery protocol

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gWiz plasmid containing luciferase (gWiz-Luc), or Green Fluorescence Protein reporter gene (gWiz-GFP) were purchased from Aldevron (Fargo, ND) as 5 mg/ml aqueous solution and used without further purification. Plasmid DNA encoding the human interferon-beta1 (IFN-β1) inserted into a pCMV6-XL4 vector was purchased from OriGene Technologies Inc. (Rockville, MD) and used without further purification. Polyethylenimine branched (PEI, molecular weight 25,000 Da) was purchased from Sigma-Aldrich (St. Louis, MO). Ethidium Bromide: 1% solution, and agarose were procured from Fisher Bioreagents (Fair Lawn, NJ). GeneRuler 1 kb (DNA ladder) and 6x DNA loading dye were purchased from Thermo Scientific (Vilnius, Lithuania). Cell Titer Glo 2.0 reagent and cell culture lysis 5x reagent were from Promega (Madison, WI). ATP, Lucifer Yellow CH dilithium, and heparin sodium salt were purchased from MP Biomedicals (Illkirch, France). Pierce BCA protein assay kit for total protein measurement was purchased from Thermo Scientific (Rockford, IL). Annexin V Alexa Fluor 647 conjugate and e-Bioscience 7-AAD viability staining solution were procured from Thermo Fisher Scientific (Carlsbad, CA). All the chemicals used for the experiments were obtained as molecular biology grade, DNase, RNase, and protease-free materials.

Dave K.M., Han L., Jackson M.A., Kadlecik L., Duvall C.L, & Manickam D.S. (2020). DNA Polyplexes of a Phosphorylcholine-Based Zwitterionic Polymer for Gene Delivery. Pharmaceutical research, 37(9), 176.

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Apoptosis Analysis of MCF-7 Cells under Normoxia and Hypoxia

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MCF-7 cells were seeded in 6 well plates at 0.3 × 10⁶ cells per well and allowed to grow overnight. 24 h normoxia, 20 h normoxia followed by 4 h hypoxia and 24 h hypoxia treatments were administered. Cells were dissociated from culture plates using 1 ml TrypLE (Thermo Scientific). Cells were washed with Annexin V binding buffer (BD Pharmingen) and stained with Annexin V Alexa Fluor 647 conjugate and SYTOX Blue at 1:1000 each (Thermo Scientific) prior to analysis using the LSR II Flow Cytometry System (BD Pharmingen).

Han J., Li J., Ho J.C., Chia G.S., Kato H., Jha S., Yang H., Poellinger L, & Lee K.L. (2017). Hypoxia is a Key Driver of Alternative Splicing in Human Breast Cancer Cells. Scientific Reports, 7, 4108.

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Quantification of Apoptosis by Annexin V

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Apoptosis was quantified using the Annexin V, Alexa Fluor™ 647 conjugate (ThermoFisher Scientific, Catalogue number A23204). Briefly, cell medium and cell pellets were collected in a fluorescence-activated cell sorting (FACS) tube. Annexin V-647 (25 μg/ml) was added to the cells. The cells were incubated for 10 min in the darkness at RT. DAPI staining solution (1 μg/ml) was then added and FACS was carried out using a BD FACS CANTO II flow cytometer.

Fafián-Labora J., Carpintero-Fernández P., Jordan S.J., Shikh-Bahaei T., Abdullah S.M., Mahenthiran M., Rodríguez-Navarro J.A., Niklison-Chirou M.V, & O’Loghlen A. (2019). FASN activity is important for the initial stages of the induction of senescence. Cell Death & Disease, 10(4), 318.

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Multimodal Cell Analysis Protocol

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Hoechst 33342 (excitation: 350 nm, emission: 461 nm) (Ref: H3570); 5,5’,6,6’–tetrachloro-1,1’,3,3’tetraethylbenzymidazolyl carbocianyne iodine (JC-1) (excitation: 488 nm, emission: 530 nm, monomer form) (excitation: 561 nm, emission: 591 nm, aggregate form) (Ref: T3168); CellRox Deep Red Reagent (excitation: 644 nm, emission: 655 nm) (Ref: C10422); Cell Event Caspase-3/7 Green Detection Reagent (excitation, 502 nm, emission: 530 nm) (Ref: C10423); Annexin V Alexa Fluor 647 conjugate (excitation: 650 nm, emission: 665 nm) (Ref: A23204); and ethidium homodimer (excitation, 528 nm, emission, 617 nm) (Ref: E1169) were purchased from ThermoFisher Scientific (Molecular Probes) (Waltham, Massachusetts, USA). Anti-phospho-Akt (Ser 473) (D9E) XP Rabbit mAb (Alexa Fluor 488 conjugate was acquired from Cell Signalling Technology (Danvers, Massachusetts, USA). Rosiglitazone, dorsomorphin, GW9662 and an Akt I-II kinase inhibitor and all other reagents unless otherwise specified were purchased from Sigma-Aldrich (Madrid, Spain).

Ortiz-Rodriguez J.M., Balao da Silva C., Masot J., Redondo E., Gazquez A., Tapia J.A., Gil C., Ortega-Ferrusola C, & Peña F.J. (2019). Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS ONE, 14(7), e0211994.

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Measuring Apoptosis After MBZ Exposure

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For measuring the fraction of cells undergoing apoptosis at different time points after MBZ exposure, 400,000 cells per well were plated in 6-well plates and allowed to adhere overnight. The next day, DMSO or MBZ was added at the indicated concentrations. At the indicated time points, the cells were removed with Accutase (Sigma), and the cell pellet was washed 1 time with ice-cold PBS. The cells were then resuspended in annexin-binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) at ~ 1 × 10⁶ cells/mL. Then 5 μL of Annexin V, Alexa Fluor 647 conjugate (ThermoFisher Scientific, A23204), or Annexin V-FITC conjugate (BioLegend) was added per 100 μL of cell suspension. Cells were incubated at room temperature for 15 minutes, and 400 μL of annexin-binding buffer was added. The cells were kept on ice until ready for analysis by flow cytometry.

Zhang L., Bochkur Dratver M., Yazal T., Dong K., Nguyen A., Yu G., Dao A., Bochkur Dratver M., Duhachek-Muggy S., Bhat K., Alli C., Pajonk F, & Vlashi E. (2018). Mebendazole Potentiates Radiation Therapy in Triple-Negative Breast Cancer. International journal of radiation oncology, biology, physics, 103(1), 195-207.

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Annexin V-Alexa Fluor 647 Apoptosis Assay

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Cells seeded in a 6-well plate were transfected with the specified plasmid for 24 hr and harvested via trypsinization in complete media to allow a 30 min recovery at 37°C. The samples were then centrifuged at 500xg for 5 min and resuspended In 150 μl of a 1:20 dilution of Annexin V-AlexaFluor 647 conjugate (Thermo Scientific #A23204). Samples were transferred to a 96-well U-bottom plate and incubated in the dark for 15 min at RT. The distribution of apoptotic cells was determined by flow cytometry on a BD LSR II.

Lam M., Marsters S.A., Ashkenazi A, & Walter P. (2020). Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress. eLife, 9, e52291.

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Quantifying Platelet Phosphatidylserine in Mice

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Male and female C57BL/6J mice (n = 6) were intravenously dosed with the following treatments: (i) saline control (equivalent volume), (ii) ML355 (1.5 mg/kg), (iii) sHDL (50 mg/kg), or (iv) ML355-sHDL (sHDL at 50 mg/kg and ML355 at 1.5 mg/kg). At different time points after administration (1, 6, and 24 hours), the phosphatidylserine exposure over time course in platelets from different groups was quantified by flow cytometry. Briefly, whole blood was collected from the saphenous vein and incubated with PE anti-mouse CD41 antibody (BioLegend, #133906) for platelet labeling and annexin V, Alexa Fluor 647 conjugate (phosphatidylserine exposure) (Thermo Fisher Scientific). The mean fluorescence intensity of annexin in platelets was quantified by flow cytometry. In addition, blood collected from mice before treatment was taken as resting platelets (negative control) and blood stimulated with PAR4-AP (200 μM) was used as activated platelets (positive control).

He H., Adili R., Liu L., Hong K., Holinstat M, & Schwendeman A. (2020). Synthetic high-density lipoproteins loaded with an antiplatelet drug for efficient inhibition of thrombosis in mice. Science Advances, 6(49), eabd0130.

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Irradiation-Induced Apoptosis Assay

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Effector cells (729.6 HTLV-1_WT or 729.6 HTLV-1_p12KO) were lethally irradiated (20 min for a dosage of 12 grays, Precision X-Ray, 320KV X-rays). Before 24, 48, and 72 h post-irradiation cells were stained with Annexin V, Alexa Fluor 647 conjugate (ThermoFisher Scientific). Briefly, cells were washed with PBS and 0.5x10⁶ cells were suspended in 1X Annexin V binding buffer (ThermoFisher Scientific). Alexa Fluor 647 conjugate (5 drops) was added to the cells and incubated at room temperature for 15 min in the dark. Cells were then washed with 2 ml of 1X Annexin V binding buffer. Cells were then collected and samples analyzed by flow cytometry (BD LSR II). Data were analyzed using FlowJo Version 10.6.

Moles R., Sarkis S., Galli V., Omsland M., Artesi M., Bissa M., McKinnon K., Brown S., Hahaut V., Washington-Parks R., Welsh J., Venzon D.J., Gutowska A., Doster M.N., Breed M.W., Killoran K.E., Kramer J., Jones J., Moniuszko M., Van den Broeke A., Pise-Masison C.A, & Franchini G. (2022). NK cells and monocytes modulate primary HTLV-1 infection. PLoS Pathogens, 18(4), e1010416.

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