Purification of Platelets from Mouse Blood

Summary

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Abstract

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Introduction

Platelets are a component of blood that functions as an initiator of blood clotting in response to damage in the blood vessel. They gather at the site of injury to plug the vessel wall¹. Platelets are anucleate fragments of cytoplasm derived from the megakaryocytes of the bone marrow under the influence of thrombopoietin and enter the circulation². They are considered as metabolically active and capable of sensing extracellular environment by activating intracellular signaling cascades that result in platelet spreading, aggregation, and hemostatic plug formation^1,3

Protocol

Mouse blood collection should be conducted with appropriate institutional animal care and use committee approval.

NOTE: The platelet purification protocol is described in a flow diagram in Figure 1.

1. Collection of Blood

1. Add 25 µL of 3.2% sodium citrate (pH 7.2) as an anti-coagulant and 0.4 mM of Gly-Pro-Arg-Pro (GPRP) in a polypropylene tube.
2. Collect approximately 200 µL of bloo.......

Discussion

Commonly, platelets are isolated by low-speed centrifugation which yields platelet-rich plasma that contains a significant number of blood cells, cellular debris, and plasma proteins which can interfere with the biochemical and physiological assays and needs further purification²¹. Therefore, it is important to use a rapid and simple method which can yield pure platelets without major contaminants. The protocol presented here describes the purification of platelets from mouse blood using a gradien.......

Materials

Name	Company	Catalog Number	Comments
APC rat anti-mouse/human CD62P (P-selectin)	Thermoscientific	17-0626-82	Platelets activation marker
Eppendorf tube	Fisher Scientific	14-222-166	Tube for centifuge
FACS DIVA software	BD Biosciences	Non-catalog item	Analysis of platelets and whole blood
FACS tube	Fisher Scientific	352008	Tubes for flow cytomtery
Fetal bovine serum (FBS)	Invitrogen	26140079	Ingredient for staining buffer
FITC rat anti-mouse CD41	BD Biosciences	553848	Platelets marker
Flow cytometer	BD Biosciences	Non-catalog item	Analysis of platelets and whole blood
FlowJo software	FlowJo, Inc.	Non-catalog item	Analysis of platelets and whole blood
Gly-Pro-Arg-Pro (GPRP)	EMD Millipore	03-34-0001	Prevent platelet clot formation
Hematocrit Capillary tube	Fisher Scientific	22-362566	Blood collection capillary tube
Hemavet	Drew Scientific	Non-catalog item	Blood cell analyzer
Hemocytometer	Hausser Scientific	3100	Cell counting chamber
Isoflurane	Baxter	1001936040	Use to Anesthetize mouse
Microscope (Olympus CKX41)	Olympus	Non-catalog item	Cell monitoring and counting
Nycodenz (Histodenz)	Sigma-Aldrich	D2158	Gradient medium
PE rat anti-mouse CD45	BD Biosciences	561087	WBC marker
PE-Cy7 rat anti-mouse TER 119	BD Biosciences	557853	RBC marker
Pipet tips 200 µL, wide-bore	ThermoFisher Scientific	21-236-1A	Transferring blood and platelet samples
Pipet tips 1000 µL, wide-bore	ThermoFisher Scientific	21-236-2C	Transferring blood and platelet samples
Phosphate buffured saline (PBS)	ThermoFisher Scientific	14040-117	Buffer for washing and dilution
Sodium chloride	Sigma-Aldrich	S7653	Physiological saline
Sodium citrate	Fisher Scientific	02-688-26	Anti-coagulant
Staining buffer	In-house	Non-catalog item	Wash and dilution buffer
Steile water	In-house	Non-catalog item	Solvent
Table top centrifuge	ThermoFisher Scientific	75253839/433607	Swinging bucket rotor centrifuge
Thrombin	Enzyme Research Laboratoty	HT 1002a	Platelet activation agonist
Tricine	Sigma-Aldrich	T0377	Buffer for Nycodenz medium