Acute 220 μm parasagittal cerebellar slices were obtained from juvenile (17–23 days old) C57BL/6 mice of both sexes, as reported previously [21 (link),39 (link),40 (link),41 (link)] (see Supplementary Materials for details), from both the vermis and hemisphere. Slices were recovered for at least 1 h in Krebs solution before being incubated for 1 h with 75 nM U46619 (Abcam, Cambridge, United Kingdom), a thromboxane agonist. For recording, slices were transferred to the recording chamber of an upright microscope (Slicescope, Scientifica Ltd., Uckfield, United Kingdom) or the HD-MEA (BioCAM X, 3Brain AG, Wädenswil, Switzerland), where Krebs solution was perfused (2 mL/min), maintained at 37 °C, and combined with 75 nM U46619. Krebs solution for slice cutting and recovery contained (in mM): 120 NaCl, 2 KCl, 1.2 MgSO4, 26 NaHCO3, 1.2 KH2PO4, 2 CaCl2, and 11 glucose, equilibrated with 95% O2-5% CO2 (pH 7.4). All drugs were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), unless otherwise specified.
U46619
Manufactured by Abcam
Sourced in United States
U46619 is a thromboxane A2 receptor agonist. It is a chemical compound used for research purposes in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Jon a antibody, by Emfret Analytics (2 mentions)
Adenosine diphosphate (adp), by Merck Group (2 mentions)
Fibrinogen, by Merck Group (2 mentions)
Fitc conjugated annexin 5, by Cell Signaling Technology (1 mentions)
Fluorescein isothiocyanate fitc conjugated anti p selectin, by Cell Signaling Technology (1 mentions)
Phycoerythrin conjugated anti cd69, by BD (1 mentions)
Fitc conjugated anti cd45, by BD (1 mentions)
Stir bars, by Chrono-Log (1 mentions)
Anti rhoa, by Cell Signaling Technology (1 mentions)
Anti arhgef1, by Cell Signaling Technology (1 mentions)
Anti g13, by Merck Group (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Thrombin, by Chrono-Log (1 mentions)
Pac 1, by BD (1 mentions)
Annexin 5, by BD (1 mentions)
Chronolume, by Chrono-Log (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
5 protocols using u46619
1
Acute Cerebellar Slice Preparation
2
Platelet Activation Assay with Vaping E-liquid
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Absolute Zero e‐liquid (18 mg nicotine, 30% propylene glycol, 70% vegetable glycerin with a menthol flavor) was obtained from The Vapor Chef (Bristol, PA). ADP, prostacyclin/prostaglandin I2 (PGI2), cotinine methanol solution, and cotinine‐D3 standard were purchased from Sigma Aldrich (St. Louis, MO). U46619 was purchased from Abcam (St. Louis, MO). Fluorescein isothiocyanate (FITC)‐conjugated anti‐P‐selectin and FITC‐conjugated Annexin V were purchased from Cell Signaling Technology, Inc (Danvers, MA). The JON/A antibody was obtained from Emfret analytics (Würzburg, Germany). The phycoerythrin‐conjugated anti‐CD69 and FITC‐conjugated anti‐CD45 were obtained from BD Biosciences (San Jose, CA). Stir bars and other disposables were purchased from Chrono‐Log Corporation (Havertown, PA). Kinetex 1.7 μm EVO C18 100 A‐LC Column 100×2.1 mm, Security Guard ULTRA holder for UHPLC Columns 2.1 to 6.4 mm, and SecurityGuard ULTRA cartridges for EVO‐C18‐UHPLC were purchased from Phenomenex Inc (Torrance, CA). Other reagents were of analytical grade.
3
Platelet Activation Signaling Pathway
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Thrombin, collagen, stir bars, and other disposables were from Chrono‐Log Corporation, whereas U46619 was purchased from Abcam. Fluorescein isothiocyanate–conjugated anti–P‐selectin, anti‐Arhgef1, anti‐actin, anti‐RhoA, anti‐pROCK, and anti‐Rap1b antibodies were purchased from Cell Signaling Technology, Inc, whereas the anti‐β3 and anti‐G13 antibodies were from Sigma Aldrich. The JON/A antibody was from Emfret analytics. Fibrinogen was purchased from Sigma Aldrich. The RhoA and Rap1b Activation Assay Biochem Kits were from Cytoskeleton Inc. Other reagents were of analytical grade.
4
Platelet aggregation and activation assay
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(S)-Duloxetine hydrochloride, ADP, and fibrinogen were purchased from Sigma Aldrich (St. Louis, MO, USA). The agonist U46619 was purchased from Abcam (Cambridge, MA, USA). Platelet aggregometry supplies such as glass cuvettes and magnetic stir bars, and luciferin-luciferase (Chrono-Lume) were purchased from the Chrono-Log Corporation (Havertown, PA, USA). Fluorescein isothiocyanate (FITC)–conjugated anti–P-selectin, PAC1, and Annexin V were purchased from BD Biosciences (San Jose, CA, USA).
5
Flow Cytometric Platelet Aggregation Analysis
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Flow cytometric analysis of platelet aggregation was conducted as recently described by Vinholt et al. [28] . In brief, platelet-rich plasma (PRP) was obtained by centrifugation at 200×g for 10 minutes and incubated with drugs. One fraction of PRP was suspended with a ratio 1:10 in dilution buffer (NaCl 134 mM, KCL 2.9 mM, MgCl2 1 mM, glucose Louis, MO, USA) and 3.1-12.5 µM U46619 (Abcam, Cambridge, UK). Unstimulated samples without agonist were analysed as negative controls.
Tubes were then shaken for 5 minutes, 1000rpm, 37°C in an Eppendorf Thermomixer Comfort (Eppendorf, Hamburg, Germany). Formalin 0.2% in PBS was added, and platelet aggregation was measured using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data was analysed and reported as described in Vinholt et al. [28] , using the FACS DIVA software (Becton Dickinson). Platelet aggregation was reported as the percentage of calcein-AM double-positive of all CV450-positive events.
Tubes were then shaken for 5 minutes, 1000rpm, 37°C in an Eppendorf Thermomixer Comfort (Eppendorf, Hamburg, Germany). Formalin 0.2% in PBS was added, and platelet aggregation was measured using a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data was analysed and reported as described in Vinholt et al. [28] , using the FACS DIVA software (Becton Dickinson). Platelet aggregation was reported as the percentage of calcein-AM double-positive of all CV450-positive events.
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