Activation of the platelets was performed using 4 μg/ml human type III collagen (Sigma-Aldrich, Israel), 4 μM ADP, or 250 μg/ml arachidonic acid (Helena Laboratories, USA). Activation was performed in PBS or FXDP (Stago, France).
Arachidonic acid
Manufactured by Helena Laboratories
Sourced in United States
Arachidonic acid is a long-chain polyunsaturated fatty acid that serves as a precursor for the biosynthesis of eicosanoids, which are signaling molecules involved in various physiological processes. It is an essential component of cell membranes and plays a crucial role in cellular function and communication.
Automatically generated - may contain errors
Lab products found in correlation
Epinephrine, by Helena Laboratories (4 mentions)
Adenosine diphosphate (adp), by Helena Laboratories (3 mentions)
Ristocetin, by Helena Laboratories (2 mentions)
Ristocetin, by Stago (1 mentions)
Annexin 5 bv421, by BD (1 mentions)
Zombie green fixable viability dye, by BioLegend (1 mentions)
Mitospy green fm, by BioLegend (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Thrombin, by Merck Group (1 mentions)
Fitc conjugated goat anti mouse igg, by ZSGB-BIO (1 mentions)
Alteplase, by Boehringer Ingelheim (1 mentions)
6 protocols using arachidonic acid
1
Platelet Activation Assays
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
Multicolor Flow Cytometry of Platelets
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BRT was purchased from Frontier Scientific Inc. (Logan, UT, USA). All phlebotomy consumables, Annexin V Binding Buffer, Stain Buffer (BSA), Compensation Beads (anti-Mouse Ig, κ/Negative Control) and anti-CD42b-APC (HIP1, 551061), anti-CD42b-PE-Cy5 (HIP1, 551141), anti-CD62P-PE (AK4 555524), anti-PAC-1-FITC (PAC1, 340507) and Annexin V-BV421 (563973) were purchased from Becton Dickinson (Brisbane, Australia). Platelet agonists adenosine diphosphate (ADP), collagen and arachidonic acid (AA) were purchased from Helena Laboratories (Melbourne, Australia) with thrombin receptor activating peptide SFLLRN (TRAP-6) purchased from Haemoview Diagnostics (Brisbane Australia). CHRONO-LUME® and all aggregation consumables were purchased from DKSH Australia (Brisbane, Australia) with MitoSOX™ Red from ThermoFisher Scientific (Brisbane, Australia). Both MitoSPY™ Green FM and Zombie Green™ Fixable Viability Dye were purchased from BioLegend (San Diego, USA). All other reagents were purchased from Sigma Aldrich (Castle Hill, Australia) unless otherwise stated.
6
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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