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 …
Declarações
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 | – |
Tags
Citar este artigo
A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets. J. Vis. Exp. (Pending Publication), e21995, doi: (2024).