Alexa fluor 488 conjugated fibrinogen

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 488-conjugated fibrinogen is a fluorescently labeled protein that can be used for various research applications. It is prepared by conjugating the Alexa Fluor 488 dye to the fibrinogen protein. Fibrinogen is a blood plasma glycoprotein that plays a crucial role in blood clotting. The Alexa Fluor 488 dye provides a green fluorescent label, allowing for the visualization and tracking of fibrinogen in biological systems.

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Lab products found in correlation

Fura 2 am 2 acetoxymethyl, by Thermo Fisher Scientific (3 mentions) Cgmp enzyme immunoassay kit, by Cayman Chemical (2 mentions) Thromboxane b2 assay kit, by Cayman Chemical (2 mentions) Thrombin, by Chrono-Log (2 mentions) Human fibrinogen, by Merck Group (2 mentions) Benzoyl peroxide, by Merck Group (2 mentions) Eclipse ti, by Nikon (1 mentions) Fitc conjugated anti p selectin, by BD (1 mentions) Phosphatidylserine, by Avanti Polar Lipids (1 mentions) Phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions) Phosphatidylcholine, by Avanti Polar Lipids (1 mentions) Anti phospho pi3k, by Cell Signaling Technology (1 mentions) Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions) Atp assay kit, by Cayman Chemical (1 mentions) Anti phospho cpla2, by Cell Signaling Technology (1 mentions) Anti phospho akt, by Cell Signaling Technology (1 mentions) Mouse monoclonal to cd62p p selectin antibody, by BioLegend (1 mentions) Antiphospho vasp, by Cell Signaling Technology (1 mentions) Anti β actin, by Cell Signaling Technology (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)

9 protocols using alexa fluor 488 conjugated fibrinogen

Visualizing Fibrin Network Structures

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Confocal microscopy was used to visualize
the fibrin networks in their native, hydrated state. For fluorescence
imaging, Alexa Fluor 488-conjugated fibrinogen (Life Technologies,
Bleiswijk, The Netherlands) was mixed with unlabeled fibrinogen in
a 1:19 molar ratio. Samples containing varying final concentrations
of fibrinogen and buffer molecules were prepared in sealed glass chambers
made of a microscope coverslip and slide with Parafilm spacers and
polymerized at 37 °C for 4 h before imaging. Imaging was performed
on a Nikon Eclipse Ti inverted microscope equipped with a 100×
oil-immersion lens (NA = 1.40), a 488 nm laser for illumination, and
a photomultiplier tube (PMT) detector. To visualize PPP clots, confocal
reflectance imaging of unlabeled samples was performed on a Zeiss
LSM510 inverted microscope with a 63× oil-immersion lens (NA
= 0.75). The samples were illuminated using a 488 nm laser, and the
reflected light was detected using a PMT detector.

Kurniawan N.A., van Kempen T.H., Sonneveld S., Rosalina T.T., Vos B.E., Jansen K.A., Peters G.W., van de Vosse F.N, & Koenderink G.H. (2017). Buffers Strongly Modulate Fibrin Self-Assembly into Fibrous Networks. Langmuir, 33(25), 6342-6352.

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Fibrinogen Adsorption Dynamics on Dextran Wafers

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Alexa Fluor 488-conjugated fibrinogen was purchased from Life Technologies (Catalog #F13191, Lot #1636855) with a DOL of 6. This was the same ligand as was used for the hMSC and dose-response experiments and was adsorbed to the PDMS stamps for 45 mins before being stamped onto the dextran-coated wafers for 5 mins. Three concentrations (30, 20 and 10 μg mL-1) were tested to create a concentration vs. fluorescent intensity curve.

Pushkarsky I., Tseng P., Warfe L., Black D., France B., Koziol-White C.J., Jester WF J.r., Trinh R.K., Lin J., Scumpia P.O., Morrison S.L., Panettieri RA J.r., Damoiseaux R, & Di Carlo D. (2018). Elastomeric sensor surfaces for high-throughput single-cell force biology. Nature biomedical engineering, 2(2), 124-137.

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Platelet Activation Assay with PAR-4 Agonist

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1 mM calcium was added to gel-filtered platelets. For flow cytometric assays, Alexa-Fluor-488–conjugated fibrinogen (Molecular Probes, Eugene, OR, USA) or FITC-conjugated anti–P-selectin (BD Pharmingen) were added with a range of concentrations of AYPGKF, a PAR-4 agonist peptide. Platelets were stimulated for 10 min at 37°C, and fixed for 10 minutes with 2% paraformaldehyde at RT. Washed platelets were diluted in Tyrode’s buffer and stained with FITC-anti-CD41, PE-anti-GPVI, and APC-anti-CD49b for 15 min before flow cytometric.

Lindsey S., Jiang J., Woulfe D, & Papoutsakis E. (2014). Platelets from mice lacking the aryl hydrocarbon receptor (AHR) exhibit defective collagen-dependent signaling. Journal of thrombosis and haemostasis : JTH, 12(3), 383-394.

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Characterization of Recombinant TFPI-α

Cited 1 time

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Commercial FXI-deficient plasma was from HRF, Inc^™ (Raleigh, NC, USA). FXI was from Haematologic Technologies, Inc (Essex Junction, VT, USA). Recombinant full-length TFPIα was produced in human embryonic kidney cells, purified on a heparin column, and characterized as described (Supplemental Figure 1).[26 (link), 27 (link)] Recombinant TF (Innovin^®) was from Siemens Healthcare Diagnostics (Newark, DE, USA). Reagents to measure thrombin generation (PPP-low, calibrator, and fluorescent substrate/CaCl₂ [FluCa]) were from Diagnostica Stago (France). CaCl₂ for clotting assays was from Sigma-Aldrich (St Louis, MO, USA). Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were from Avanti Polar Lipids (Alabaster, AL, USA). Large unilamellar phospholipid vesicles (41% Phosphatidylcholine/44% phosphatidylethanolamine/15% phosphatidylserine) were made as described.[14 (link)] Recombinant tissue plasminogen activator (tPA) was from American Diagnostica, Inc (Stamford, CT, USA). AlexaFluor488-conjugated fibrinogen was from Molecular Probes (Eugene, OR, USA). Control isotype IgG antibodies were from Affinity Biologicals (Ontario, Canada).

Reitsma S.E., Holle L.A., Bouck E.G., Monroe D.M., Mast A.E., Burthem J., Bolton-Maggs P.H., Gidley G.N, & Wolberg A.S. (2022). Tissue Factor Pathway Inhibitor Is a Potential Modifier of Bleeding Risk in Factor XI Deficiency. Journal of thrombosis and haemostasis : JTH, 21(3), 467-479.

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Mulberroside C Signaling Pathway Analysis

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Mulberroside C (Figure 1) was provided by ChemFaces (Wuhan, China). Mouse monoclonal to CD62P (P-selectin) antibody was purchased from Biolegend (San Diego, CA, USA). Fura 2-AM (2-acetoxymethyl) and Alexa Fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). The BCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA). The serotonin ELISA kit was provided by Labor Diagnostika Nord GmbH and Co. (Nordhorn, Germany). Collagen, U46619, and thrombin were purchased from Chrono-Log Co. (Havertown, PA, USA). The thromboxane B₂ assay kit, cAMP, cGMP enzyme immunoassay kit, and ATP assay kit were obtained from Cayman Chemical (Ann Arbor, MI, USA). Cell signaling (Beverly, MA, USA) supplied antiphospho-p38^MAPK, antiphospho-ERK (1/2), antiphospho-VASP (Ser¹⁵⁷), antiphospho-VASP (Ser²³⁹), antiphospho-cPLA₂ (Ser⁵⁰⁵), antiphospho-PI3K (Tyr⁴⁵⁸), antiphospho-Akt (Ser⁴⁷³), antiphospho-inositol-3-phosphate receptor type I (Ser¹⁷⁵⁶), antiphospho-PLC_γ₂ (Tyr⁷⁵⁹), anti-β-actin, and antirabbit secondary antibodies.

Kwon H.W., Lee D.H., Rhee M.H, & Shin J.H. (2021). In Vitro Antiplatelet Activity of Mulberroside C through the Up-Regulation of Cyclic Nucleotide Signaling Pathways and Down-Regulation of Phosphoproteins. Genes, 12(7), 1024.

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Platelet Activation Pathway Analysis

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Collagen, ADP, and thrombin were obtained from Chrono-Log Co. (Collagen cat # 385 ADP cat #384 thrombin cat #386, CHRON-LOG Corporation, Havertown, PA, USA). Paraformaldehyde (CAS no. 30525-89-4) and glutaraldehyde (CAS no. 111-30-8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ATP Assay kit was obtained from the Biomedical Research Service (REF. 373, BMR, 3434 Main St, Buffalo, NY, USA). thrombin from human plasma was purchased from Sigma-Aldrich (Lot # SLCF9776 CAS no. 9002-04-4 647-014-00-9). Fura 2-AM (2-acetoxymethyl) (CAS number: 108964-32-5) and Alexa Fluor 488-conjugated fibrinogen (Cat # A32723) were obtained from Invitrogen (Eugene, OR, USA). All antibodies were supplied by Cell Signaling (Beverly, MA, USA).

Akram A.W., Saba E, & Rhee M.H. (2024). Antiplatelet and Antithrombotic Activities of Lespedeza cuneata via Pharmacological Inhibition of Integrin αIIbβ3, MAPK, and PI3K/AKT Pathways and FeCl3-Induced Murine Thrombosis. Evidence-based Complementary and Alternative Medicine : eCAM, 2024, 9927160.

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Fibrinolysis Assay Protocol

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Human fibrinogen, thrombin, FXa, FXIIIa, urokinase-type plasminogen activator (u-PA), trizma base, and trizma HCl were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paranitroaniline chromogenic substrates were obtained from Chromogenix (Milan, Italy). Alexa Fluor 488-conjugated fibrinogen was purchased from Invitrogen (Eugene, OR, USA). Other reagents used were of analytical grade and purchased from commercial sources.

Choi J.H, & Kim S. (2022). In Vitro Antithrombotic, Hematological Toxicity, and Inhibitor Studies of Protocatechuic, Isovanillic, and p-Hydroxybenzoic Acids from Maclura tricuspidata (Carr.) Bur. Molecules, 27(11), 3496.

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Fibrinogen-Conjugated Nanoparticle Synthesis

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Alexa Fluor 488 – conjugated fibrinogen was purchased from Invitrogen. Human fibrinogen, thrombin and gold nanorods (GNRs) (axial diameter: 9.0–11.0 nm; longitudinal diameter: 36.9–45.1 nm), methyl methacrylate (MMA), butylmethacrylate (BMA), methylbenzoate, polyethylene glycol 400, benzoyl peroxide and N,N-dimethyl-p-toluidine were procured from Sigma, while methylene blue, benzoyl peroxide and Drabkin's solution were products of Merck, Thomas baker and Linear Chemicals S.L.U., respectively. The rest of the chemicals were either from Sigma or Merck. All reagents were of analytical grade. Type I deionized water (18.2 MΩ cm, Millipore) has been used throughout the experiment.

Singh N., Kulkarni P.P., Tripathi P., Agarwal V, & Dash D. (2024). Nanogold-coated stent facilitated non-invasive photothermal ablation of stent thrombosis and restoration of blood flow. Nanoscale Advances, 6(5), 1497-1506.

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Platelet Activation and Signaling Assays

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Derrone was purchased from ChemFaces (Wuhan, China). Collagen was purchased from Chrono-Log Co. (Havertown, PA, USA). Fura 2-AM (2-acetoxymethyl) and Alexa Fluor 488-conjugated fibrinogen were obtained from Invitrogen (Eugene, OR, USA). Serotonin ELISA kit was purchased from Labor Diagnostika Nord GmbH and Co. (Nordhorn, Germany). Bicinchoninic acid protein assay kit was purchased from Pierce Biotechnology (IL, USA). Cayman chemical (Ann Arbor, MI, USA) offered thromboxane B₂ assay kit, cAMP, cGMP enzyme immunoassay kit, and U46619. Cell Signaling (Beverly, MA, USA) supplied all antibodies. The adhesion kit (fibronectin coated) was obtained from Cell Biolabs (San Diego, CA, USA).

Shin J.H., Irfan M., Rhee M.H, & Kwon H.W. (2021). Derrone Inhibits Platelet Aggregation, Granule Secretion, Thromboxane A2 Generation, and Clot Retraction: An In Vitro Study. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 8855980.

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