Desiccated B. moojeni venom was purchased from Bioagents Serpentarium (Batatais, SP, Brazil). Acetonitrile, acrylamide, ammonium bicarbonate, ammonium persulphate, bromophenol blue, bovine fibrinogen, glycine, β-mercaptoethanol, N, N′-methylene-bis-acrylamide, sodium dodecyl sulphate (SDS), N, N, N′, N′-tetramethylethylenediamine (TEMED), trifluoroacetic acid, and Tris were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Molecular mass markers for electrophoresis and all chromatographic media (DEAE-Sephacel, Sephadex G-75, and C2/C18 columns) were purchased from GE Healthcare Technologies (Uppsala, Sweden). All the agonists used in the platelet aggregation assays (collagen, adenosine diphosphate, epinephrine, and ristocetin) were purchased from Helena Laboratories (Beaumont, Texas, USA). All other reagents used were of analytical grade.
Epinephrine
Manufactured by Helena Laboratories
Sourced in United States
Epinephrine is a laboratory equipment product used to measure the presence and concentration of the hormone epinephrine, also known as adrenaline, in biological samples. It serves as a tool for researchers and clinicians to analyze and quantify epinephrine levels in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Arachidonic acid, by Helena Laboratories (4 mentions)
Adenosine diphosphate (adp), by Helena Laboratories (4 mentions)
Ristocetin, by Helena Laboratories (3 mentions)
Thrombin, by Merck Group (2 mentions)
Fitc conjugated goat anti mouse igg, by ZSGB-BIO (2 mentions)
Ammonium persulphate, by Merck Group (1 mentions)
Bovine fibrinogen, by Merck Group (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
N methylenebisacrylamide, by Merck Group (1 mentions)
Sodium dodecyl sulphate (sds), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Deae sephacel, by GE Healthcare (1 mentions)
Sephadex g 75, by GE Healthcare (1 mentions)
C2 c18 columns, by GE Healthcare (1 mentions)
Ristocetin, by Stago (1 mentions)
Tnf α, by R&D Systems (1 mentions)
7 protocols using epinephrine
1
Proteomic Analysis of B. moojeni Venom
2
Platelet aggregation assay
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Diluted PRP (3 × 108 plt/ml in platelet-poor plasma) was placed in an aggregometer cuvette at 37°C and stirred. Agonists were added (ADP, arachidonic acid, and epinephrine were purchased from Helena Laboratories; collagen was purchased from Bio/Data Corporation; TRAP-14 was obtained from Polypeptide group; and ristocetin was obtained from Stago) at concentrations given in the text. Light transmission was recorded on an APACT 4004 optical aggregation system (Labor BioMedical Technologies GmbH).
3
Platelet Aggregation Profiler Analysis
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LTA was performed on the PEP-8E Platelet Aggregation Profiler (Ref: 106077, Bio/Data Corporation, Horsham, USA) according to the manufacturer’s recommendations. Platelet counts were not adjusted for the PRP prior to analysis. Aggregation was stimulated by the addition of the agonists ADP (Ref: 5366, Helena Laboratories, Beaumont, USA), collagen (COLtest, Ref: 06675832, Roche Products, Randburg, South Africa), arachidonic acid (Ref: 5364, Helena Laboratories, Beaumont, USA), epinephrine (Ref: 5367, Helena Laboratories, Beaumont, USA), and ristocetin (Ref: 5199, Helena Laboratories, Beaumont, USA). The final agonist concentrations were based on the CSLI guidelines [11 ]. The final concentration for each agonist was as follows: ADP (2 μM), collagen (2 μg/mL), arachidonic acid (500 μg/mL), epinephrine (5 μM), and ristocetin (1.2 mg/mL). Light absorbance was measured for 6 min following agonist addition. We reported the maximum aggregation (MA), primary slope (PS), and area under the curve (AUC) for each sample. The CLSI guideline recommend the use of MA and slope in reporting of LTA results; however, AUC is only reserved for reporting of impedance aggregometry results [11 ].
4
Platelet Activation Assay Reagents
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Arachidonic acid, ADP, and epinephrine were purchased from Helena Laboratories. For Arachidonic acid (ref 5364), sodium arachidonate was lyophilized at 5 mg/mL and reconstituted in molecular grade water to a stock concentration of 1,600 μmol/L as per manufacturer recommendations. For adenosine diphosphate (ref 5366), adenosine 5-diphosphate was reconstituted to a concentration of 200 μmol/L and diluted to concentrations of 20 μmol/L, 5 μmol/L, and 1 μmol/L. For epinephrine (ref 5367), L-epinephrine bitartrate was reconstituted to a concentration of 3 mmol/L and diluted to 10 μmol/L, 0.4 μmol/L, and 0.1 μmol/L concentrations, which have been previously published.28 (link) Thrombin (0020301100), used in flow cytometry, was purchased from HemosIL lyophilized from bovine thrombin at 35 U/mL with bovine albumin, calcium chloride, buffer, and stabilizers, and used at a diluted concentration of 0.025 units.
5
Inducing Platelet Activation and Inflammation
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To induce platelet activation, the mice were given an intraperitoneal injection of 75 μg/kg collagen (Nycomed Pharma) and 30 μg/kg epinephrine (Helena Laboratories) 3 min before euthanasia.19 (link),20 (link) To increase inflammation, 250 ng TNF-α (RD Systems) were injected 4 h before the animals were sacrificed. After euthanasia, intracardiac puncture was performed and phosphate-buffered saline was perfused for 3 min, followed by 10% formalin for another 3 min. Where noted, 25 mg P-selectin blocking antibody were injected 4 h prior to euthanasia.
6
Platelet Aggregation Inhibition by Platycodin D
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Platycodin D (PD) was purchased from Fluka (Steinheim, Germany) with a purity ≥ 99%. Collagen, adenosine diphosphate (ADP), arachidonic acid and epinephrine were from Helena laboratories (Beaumont, Texas, USA). Thrombin was purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated mouse anti-human CD41a and PAC-1 antibody were from BD Biosciences (San Jose, CA, USA) and Becton–Dickinson (San Jose, CA, USA) respectively. PE-conjugated anti-human/mouse CD62p (P-Selectin) and anti-human Glycoprotein VI purified antibody were purchased from eBioscience (San Diego, CA, USA). FITC-conjugated anti-CD42b antibody was from Abcam (Cambridge, MA, USA). FITC-conjugated goat anti-mouse IgG was purchased from ZSGB-BIO (Beijing, China). β-actin antibody and anti-rabbit IgG (HRP-linked) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Platelet Function Analysis Protocol
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Alteplase was purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany). Adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid and epinephrine were purchased from Helena Laboratories (Beaumont, TX, USA). Collagen-related peptide (CRP) was prepared as previously described.17 (link) Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD41a and FITC-conjugated mouse anti-human PAC-1 antibodies were purchased from BD Biosciences (San Jose, CA, USA) and Becton Dickinson (San Jose, CA, USA), respectively. Phycoerythrin (PE)-conjugated mouse anti-human CD62p (P-selectin) and purified mouse anti-human glycoprotein (GP) VI antibody were purchased from eBioscience (San Diego, CA, USA). FITC-conjugated mouse anti-human CD42b antibody was purchased from Abcam (Cambridge, MA, USA). FITC-conjugated goat anti-mouse IgG was purchased from ZSGB-BIO (Beijing, China). Thrombin was purchase from Sigma-Aldrich (St Louis, MO, USA).
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