A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets

Published: February 29, 2024

Abstract

Source: Molica, F. et al., Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation. J. Vis. Exp. (2017)

This video demonstrates turbidimetric analysis to assess collagen-induced platelet aggregation in pre-treated, washed human platelets. Control wells show collagen-induced aggregation, whereas a high concentration of inhibitors blocks Pannexin-1 channels, thereby inhibiting collagen-induced platelet aggregation,  evident from unchanged turbidity.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation Prepare a 100 mL aqueous solution of acid-citrate-dextrose (ACD) by dissolving 1.4 g citric acid monohydrate (C6H8O7•H2</su…

Representative Results

Figure 1: Aggregometry. (A) Representative image of glass cuvettes (containing a stirring magnet) used for aggregometry. The cuvette on the left shows resting platelets while the cuvette on the right illustrates platelet aggregates after collagen-induced activation. (B) Representative image of an 8-well aggregometer for turbidimetric measurements. The wells …

Açıklamalar

The authors have nothing to disclose.

Materials

Citric acid monohydrate (C6H8O7*H2O) Roth 5949-29-1 danger of eye damage/irritation
Trisodium citrate dihydrate (Na3C6H5O7*2H2O) Sigma-Aldrich S1804
D(+)-glucose Sigma-Aldrich G8270
Sodium chloride (NaCl) Sigma-Aldrich S9888
Potassium chloride (KCl) Sigma-Aldrich P9541
Sodium bicarbonate (NaHCO3) Sigma-Aldrich S6014
Sodium dihydrogenophosphate monohydrate (NaH2PO4*H2O) Sigma-Aldrich S9638
Magnesium chloride hexahydrate (MgCl2*6H2O) Sigma-Aldrich M9272
Calcium chloride hexahydrate (CaCl2*6H2O) Sigma-Aldrich 442909 danger of eye damage/irritation
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes) ThermoFisher Scientific 15630
Human serum albumin CSL Behring 00257/374
Hydrochloric acid Sigma-Aldrich 320331 Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages.
Eppendorf 5810 R Fisher Scientific
Heparin Drosspharm AG/SA 20810
Prostacyclin I2 (PGI2) Cayman 18220
Apyrase from potatoes Sigma-Aldrich A6535
Fibrinogen (Haemocomplettan) CSL Behring HS 73466011
Thrombo-aggragometer SD-Medical SD-Innovation TA8V
Brilliant blue FCF (Erioglaucine disodium salt) Sigma-Aldrich 80717 Harmful to aquatic life with long lasting effects (Avoid release to the environnement)
Collagen Horm, Nycomed
Arachidonic acid Bio/Data corporation C/N 101297
Cell counter Sysmex KX-21N Sysmex Digitana
HEPES Gibco 15630-056
Glass cuvettes SD-Innovation THCV1000
Magnetic stirrers SD-Innovation THA100

Etiketler

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A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets. J. Vis. Exp. (Pending Publication), e21995, doi: (2024).

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