Alexa 488 conjugated fibrinogen

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa 488-conjugated fibrinogen is a fluorescently labeled protein that can be used for various research applications. Fibrinogen is a plasma glycoprotein that plays a crucial role in blood coagulation, and the Alexa 488 dye provides a fluorescent label for detection and visualization purposes. This product can be utilized in cell biology, biochemistry, and other related research fields, but a detailed description of its intended use is not provided to maintain an unbiased and factual approach.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Calcineurin cellular assay kit, by Enzo Life Sciences (1 mentions) Adenosine diphosphate (adp), by Helena Laboratories (1 mentions) Pe conjugated jon a antibody, by Emfret Analytics (1 mentions) Facscalibur, by BD (1 mentions) Tcs sp5 inverted microscope, by Leica (1 mentions) Poly l lysine coated coverslips, by BD (1 mentions) Hcx pl apo cs 63 1.40 0 60 oil objective, by Leica (1 mentions) Nis elements ar 3, by Nikon (1 mentions) Rhodamine b isothiocyanate dextran, by Merck Group (1 mentions)

Close spelling variants of this product

Alexa Fluor 488–conjugated human fibrinogen Alexa Fluor 488-conjugated fibrinogen Alexa 488 Fibrinogen

7 protocols using alexa 488 conjugated fibrinogen

Neutrophil Fibrinogen Binding Assay

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DIV neutrophils (1 × 10⁵ cells/well) in HBSS+ were plated onto 96-well 4HBX plates coated with 10% FBS, 25μg/ml TDB, 10μg/mlfurfurman, or 100μg/ml curdlan and the plate was spun at 250 x g for 2 min. Negative controls were treated with 2.5mM EDTA for 30 min at 37°C and plated in wells coated with 10% FBS. Cells were incubated at 37°C in the presence of 5% CO₂ for 20 min. 30 μg/mL Alexa 488-conjugated fibrinogen (Invitrogen) was added to the wells, and the cells were incubated at 37°C in presence of 5% CO₂ for an additional 20 min. In some FBS-coated wells, 2.5mM manganese (Mn²⁺) was added to cells for the last 15 min of incubation. Following stimulation, the supernatants were removed, and the wells were washed twice with cold PBS. 200μL of cold 2.5 mM EDTA in PBS without Ca²⁺ and Mg²⁺ was then added to each well. The cells were incubated at 4°C for an hour to promote lifting of the cells and transferred into 5 mL FACS tubes with 1% formaldehyde. The FACS tubes were covered with paraffin, and stored in the dark, at 4°C until flow cytometry analysis. The percentage of fibrinogen positive cells were normalized to EDTA-treated level and presented as a percentage of Mn²⁺-treated level. Data were compiled from n = 4–5 independent experiments in technical duplicate, presented as mean ± SEM, and assessed using two-way ANOVA with Sidak's post-test.

Nguyen G.T., Xu S., Adams W., Leong J.M., Bunnell S.C., Mansour M.K., Sykes D.B, & Mecsas J. (2021). Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation. iScience, 24(8), 102871.

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Platelet Activation Regulation by Calcineurin

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ADP and collagen were purchased from Helena Laboratories (Beaumont, TX), while thrombin was from Hematologic Technologies Inc. (Essex Junction, VT). Protease activated receptor 4 –activating peptide (PAR4-AP) AYPGKF was synthesized by the Protein Core at Baylor College of Medicine (BCM). CsA and FK506 were from Calbiochem-EMD Millipore (Darmstadt, Germany). Alexa 488-conjugated fibrinogen was from Invitrogen-life Technologies (Grand Island, NY). Human fibrinogen was from Enzyme Research Laboratories Inc. (South bend, IN). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD62 (P-selectin), Phycoerythrin (PE)–conjugated anti-mouse CD41 (αIIb) and control IgG tagged to PE or FITC were purchased from BD Bioscience (San Jose, CA). PE-conjugated JON/A antibody was from Emfret Analytics (Eibelstadt, Germany). Antibodies to phospho Src Tyr⁴¹⁸, Src, phospho p38 (Thr¹⁸⁰ Tyr¹⁸²), p38, phospho filamin (Ser²¹⁵²), and filamin were from Cell Signaling Technologies (Beverly, MA). Antibodies to PP2B-Aα, PP2B-Aβ were from Santa Cruz Biotechnology (Santa Cruz, CA). Calcineurin cellular assay kit was from Enzo Life Sciences, Inc. (Farmingdale, NY). A preformed mix of four independent (SMART pool) siRNAs targeting human PP2B-Aβ and non-specific control siRNA pool were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO).

Khatlani T., Pradhan S., Da Q., Gushiken F.C., Bergeron A.L., Langlois K.W., Molkentin J.D., Rumbaut R.E, & Vijayan K.V. (2014). The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside-in αIIbβ3 integrin signaling. Journal of thrombosis and haemostasis : JTH, 12(12), 2089-2101.

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Partial Agonist Primes αVβ3 Receptor

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Partial agonists of αVβ3 prime the receptor to bind the fibrinogen by inducing the receptor to adopt a high-affinity, ligand-binding conformation, whereas pure antagonists do not.^{33 (link)} HEK-αVβ3 cells were washed, resuspended in HBMT containing 1 mM MgCl₂ and 2 mM CaCl₂ at 2 × 10⁶ mL, and either left untreated (control) or incubated with 100 μM RGDS, or the test compound at 10 μM for 20 min at room temperature. The samples were then fixed with 4% paraformaldehyde in Dulbecco’s phosphate-buffered saline (PBS) for 40 min at room temperature, followed by quenching of the reaction with 5 mM glycine for 5 min at room temperature. After the cells were washed with HBMT, they were resuspended in HBMT containing 1 mM MgCl₂ and 2 mM CaCl₂. Alexa⁴⁸⁸-conjugated fibrinogen was then added (Invitrogen), and the mixture was incubated for 30 min at 37 °C. The cells were then washed and analyzed by flow cytometry (FACSCalibur; Becton Dickinson).

Sen S., Spasic A., Sinha A., Wang J., Bush M., Li J., Nešić D., Zhou Y., Angiulli G., Morgan P., Salas-Estrada L., Takagi J., Walz T., Coller B.S, & Filizola M. (2022). Structure-Based Discovery of a Novel Class of Small-Molecule Pure Antagonists of Integrin αVβ3. Journal of chemical information and modeling, 62(22), 5607-5621.

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Staphylococcus aureus Fibrinogen Binding

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S. aureus strains Reynolds (CP5 and CP^-) (mCherry-labeled, 1*10⁸/ml) were pre-incubated with human plasma (3%) for 30 min at 37°C in Veronal buffer containing 5 mM CaCl₂ and 2.5 mM MgCl₂ (VBS⁺⁺) to deposit C3b on the bacterial surface. After a washing step with VBS++-0.5% BSA, bacteria were incubated with Efb or Efb mutants (0.5 μM) for 1 hour at 37°C, shaking (600 rpm). Following another washing step, a 1 hour incubation with Alexa-488 conjugated fibrinogen (60 µg/ml, Invitrogen) at 37°C shaking was performed, after which bacteria were fixed with formaldehyde (1%). For visualization by confocal microscopy, samples were transferred onto poly-L-lysine coated cover slips (0.45 µm; 12 mm diameter; Becton Dickinson) or as a control, samples were analyzed by flow cytometry. Confocal images were acquired using a Leica TCS SP5 inverted microscope equipped with HCX PL APO CS 63×/1.40–0.60 OIL objective (Leica Microsystems, The Netherlands).

Kuipers A., Stapels D.A., Weerwind L.T., Ko Y.P., Ruyken M., Lee J.C., van Kessel K.P, & Rooijakkers S.H. (2016). The Staphylococcus aureus polysaccharide capsule and Efb-dependent fibrinogen shield act in concert to protect against phagocytosis. Microbiology, 162(7), 1185-1194.

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Neutrophil Adhesion and Fibrinogen Binding

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DIV neutrophils (1 x 10 5 cells/well) in HBSS+ were plated onto 96-well 4HBX plates coated with 10% FBS, 25g/ml TDB, 10g/ml furfurman, or 100g/ml curdlan and the plate was spun at 250 x g for 2 minutes. Negative controls were treated with 2.5mM EDTA for 30 minutes at 37C and plated in wells coated with 10% FBS. Cells were incubated at 37°C in the presence of 5% CO2 for 20 minutes. 30 g/ml Alexa 488-conjugated fibrinogen (Invitrogen) was added to the wells, and the cells were incubated at 37°C in presence of 5% CO2 for an additional 20 minutes. In some FBS-coated wells, 2.5mM manganese (Mn 2+ ) was added to cells for the last 15 minutes of incubation. Following stimulation, the supernatants were removed, and the wells were washed twice with cold PBS. 200l of cold 2.5 mM EDTA in PBS without Ca 2+ and Mg 2+ was then added to each well. The cells were incubated at 4C for an hour to promote lifting of the cells and transferred into 5 ml FACS tubes with 1% formaldehyde. The FACS tubes were covered with paraffin, and stored in the dark, at 4C until flow cytometry analysis. The percentage of fibrinogen positive cells were normalized to EDTA-treated level and presented as a percentage of Mn 2+ -treated level.

Nguyen G.T., Xu S., Bunnell S.C., Mansour M.K., Sykes D.B., & Mecsas J. (2021). Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin andCandidastimulation.

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Visualizing Platelet Adhesion in Brain

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Coronal brain cryosections (10 µm) were incubated with 20 µg/mL Alexa 488-conjugated fibrinogen (Thermo Fisher Scientific) for 2 h before fixing in 4% paraformaldehyde. Platelets were stained using rat anti-mouse GPIbα (1:100) and Alexa 555-conjugated goat anti-rat IgG. Nuclei were counterstained with DAPI (1:1000).

Robador J.R., Feinauer M.J., Schneider S.W., Mayer F.T., Gorzelanny C., Sacharow A., Liu X., Berghoff A., Brehm M.A., Hirsch D., Stadler J., Vidal-y-Si S., Wladykowski E., Asong M., Nowak K., Seiz-Rosenhagen M., Umansky V., Mess C., Pantel K., Winkler F, & Bauer A.T. (2021). Involvement of platelet-derived VWF in metastatic growth of melanoma in the brain. Neuro-oncology Advances, 3(1), vdab175.

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Intravital Imaging of Fibrin Formation

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Intravital microscopy was performed to analyze fibrin formation in vivo. Briefly, Rhodamine B isothiocyanate-dextran (5 mg/body; Sigma Aldrich, St. Louis, MO, USA), Hoechst 33342 (3 mg/body; Thermo Fisher Scientific), and Alexa 488-conjugated fibrinogen (300 μg/body; Thermo Fisher Scientific) were injected into anesthetized mice. Sequential images of testicular vein (at least 80 µm diameter) were obtained using a resonance scanning confocal microscope (Nikon A1RNP; Nikon, Tokyo, Japan) after local endothelial disruption induced by laser irradiation (wavelength 700 nm). The signal intensity of fibrin formation (shown by Alexa 488-conjugated fibrinogen signals) was quantified using NIS-Elements AR 3.2 (Nikon).

Ohmori T., Nagao Y., Mizukami H., Sakata A., Muramatsu S.I., Ozawa K., Tominaga S.I., Hanazono Y., Nishimura S., Nureki O, & Sakata Y. (2017). CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice. Scientific Reports, 7, 4159.

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