Purification and Activation of Platelets from Murine Whole Blood

Published: August 31, 2023

Abstract

Source: Narciso, M. G., et al. Purification of Platelets from Mouse Blood. J. Vis. Exp. (2019).

In this video, we describe a protocol for purifying platelets from murine whole blood and their subsequent activation with the platelet agonist thrombin. When analyzed with flow cytometry, the purified resting platelets are characterized based on the expression of their cell-surface marker CD41, whereas purified activated platelets co-express both the surface markers CD41 and CD62P.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Platelet Purification NOTE: Platelet purification should be carried out at room temperature to prevent their degradation. Make sure that the temperature of the reagents and instruments is between 18 to 22 °C. To prevent platelet degradation, fast pipetting, and vigorous shaking should…

Representative Results

Figure 1. Mouse blood before and after centrifugation with Iohexol gradient medium. (A) Iohexol and blood form two separate layers before centrifugation if the blood sample is added carefully. Left panel shows schematic diagram and right panel shows the actual image. (B) Four separate layers are seen after centrifugation of whole blood with gradient medium. Left panel shows schematic diagram of …

開示

The authors have nothing to disclose.

Materials

APC rat anti-mouse/human CD62P
(P-selectin)
Thermoscientific  17-0626-82  Platelets activation marker
Eppendorf tube Fisher Scientific 14-222-166 Tube for centrifuge
Fetal bovine serum (FBS)  Invitrogen  26140079 Ingredient for staining buffer
FITC rat anti-mouse CD41  BD Biosciences  553848 Platelets marker
Gly-Pro-Arg-Pro (GPRP) EMD Millipore 03-34-0001 Prevent platelet clot formation
Pipet tips 1000 μL, wide-bore ThermoFisher Scientific  21-236-2C  Transferring blood and platelet samples
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
Phosphate-buffered saline (PBS) ThermoFisher Scientific 14040-117 Buffer for washing and dilution
Staining buffer In-house Non-catalog item Wash and dilution buffer
Thrombin Enzyme Research Laboratoty HT 1002a Platelet activation agonist
Sodium chloride  Sigma-Aldrich  S7653  Physiological saline
Table top centrifuge  ThermoFisher Scientific  75253839/433607  Swinging bucket rotor centrifuge
Tricine  Sigma-Aldrich  T0377  Buffer for Nycodenz medium
FACS tube  Fisher Scientific  352008 Tubes for flow cytometry

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記事を引用
Purification and Activation of Platelets from Murine Whole Blood. J. Vis. Exp. (Pending Publication), e21561, doi: (2023).

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