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 |
