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3 protocols using dade innovin lipidated tissue factor tf

1

Multicolor Flow Cytometry of Platelet Activation

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Reagents were obtained as follows: Alexa Fluor (AF) 647 conjugated anti-human CD41 (Bio-Rad, Raleigh, NC, USA), Anti-human CD18 antibody (clone TS1/18), PE anti-human CD11a (BioLegend, San Diego, CA, USA), AF647 conjugated human fibrinogen (ThermoFisher Scientific, Grand Island, NY, USA), Sytox-green (Life Technologies, Grand Island, NY, USA), fibrillar collagen (type I, Chrono-log, PA, USA), Dade Innovin lipidated tissue factor (TF, Siemens, Malvern, PA, USA), D-Phe-Pro-Arg-CMK (PPACK, Santa Cruz Biotechnology, Dallas, TX, USA), corn trypsin inhibitor (CTI, Haematologic Technologies, Essex Junction, VT, USA), tPA (Abcam, Cambridge, MA, USA), H-Gly-Pro-Arg-Pro-OH acetate salt (GPRP, Bachem Americas, Torrance, CA, USA), ε-aminocaproic acid (εACA), ethylenediaminetetraacetic acid (EDTA), and Sigmacote (Sigma, St. Louis, MO, USA).
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2

Platelet Activation and Thrombosis Assays

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Reagents were obtained as follows: anti-human CD61 antibody (BD Biosciences, San Jose, CA. Cat#: 555754), Alexa Fluor 647–conjugated human fibrinogen (Life Technologies, Grand Island, NY. Cat#: F35200), Alexa Fluor 488-conjugated annexin V (ThermoFisher Scientific, Waltham, MA. Cat#: A13201), collagen (type I; Chrono-Log, Havertown, PA. Cat#: 385), Dade Innovin lipidated tissue factor (TF, Siemens, Malvern, PA, USA), Sigmacote® (Millipore Sigma, Burlington, MA. Cat#: SL2-100ML), Phe-Pro-Arg-chloromethylketone (PPACK, Haematologic Technologies, Essex Junction, VT. Cat#: FPRCK-01), corn trypsin inhibitor (CTI, Haematologic Technologies, Essex Junction, VT, USA), Pam3CKS4 (NOVUS Biologicals, CO. Cat#: NBP2-25297), MALP-2 (NOVUS Biologicals, CO. Cat#: NBP2-26219), Polyinosinic-polycytidylic acid HMW (NOVUS Biologicals, CO. Cat#: NBP2-25288), LPS (NOVUS Biologicals, CO. Cat#: NBP2-25295), Imiquimod (NOVUS Biologicals, CO. Cat#: NBP2-26228), CpG oligodeoxynucleotides (NOVUS Biologicals, CO. Cat#: NBP2-26232), Vesatolimod (MedChemExpress, NJ. Cat#: HY-15601), GSK2245035 (MedChemExpress, NJ. Cat#: HY-118250), Bay 11-7082 (Millipore Sigma, MO. Cat#: B5556), and IKK inhibitor VII (Millipore Sigma, MO. Cat#: 401486).
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3

Evaluating Platelet Function and Coagulation

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Reagents were obtained as follows: Alexa Fluor 488 conjugated anti‐human CD61 (Bio‐Rad), AF647 conjugated human fibrinogen (ThermoFisher Scientific), type I fibrillar collagen (Chrono‐log), Dade Innovin lipidated tissue factor (TF, Siemens), CTI and FXIa (Haematologic Technologies), 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES; Fisher Scientific), Sigmacote (Sigma), Anti‐FVIII (4A4) and anti‐FIX (BC2; Green Mountain Antibodies), Anti‐TFPI (gA200 hIgG1, Bayer, Whippany), bispecific antibody (emicizumab), reFIX‐V181T variant,25 and a recombinant rIgG4 containing complementarity‐determining regions originally derived from a patient (NB41, as described in van den Brink26).
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