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

Samenvatting

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

1. Prepare a 100 mL aqueous solution of acid-citrate-dextrose (ACD) by dissolving 1.4 g citric acid monohydrate (C₆H₈O₇•H₂O, 66.6 mM), 2.5 g trisodium citrate dihydrate (Na₃C₆H₅O₇•2 H₂O, 85 mM) and 2 g of anhy....

Resultaten

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 co.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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	-