Purification and Activation of Platelets from Murine Whole Blood

Overview

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 be avoided during the procedure.

1. Add 600 µL of iohexol gradient medium (12% iohexol powder in 0.85% sodium chloride, 5 mM Tricine, pH 7.2) into a 1.5 mL tube.
2. Add 200 µL of blood sample slowly down the side of the tube on top of the gradient medium with a wide bore pipette tip so that blood and iohexol medium do not mix (Figure 1A).
3. Centrifuge the sample containing tube at 400 x g for 20 min at 20 °C in a swinging bucket rotor with slow acceleration and deceleration.
4. Collect most of the platelet-rich layer and a small fraction of the platelet-poor layer (total 300 to 400 µL) using a wide bore pipette tip without disturbing the RBC and WBC layers (Figure 1B). Transfer the platelet sample to a new tube.
   NOTE: If the platelet count is less in the blood or a smaller volume of blood is used, the platelet-rich layer and platelet-poor layer can be seen as a single layer.
5. Add 1 mL of PBS to the platelet sample, mix by inverting, and centrifuge at 800 x g for 10 min at 20 °C in a swinging bucket rotor. Discard the solution completely and add 200 µL of PBS to resuspend the platelets with mild pipetting.
   NOTE: This step is optional, as it removes the gradient medium and plasma proteins and increases the sensitivity of platelets to agonists such as thrombin. Since different centrifuges have different programs, the slow acceleration/deceleration can be determined by reading the user manual of the centrifuge.
6. Transfer 30 µL of this sample into a new tube to count platelets. The remaining platelets can be used for biochemical and physiological assays such as platelet activation, aggregation, granule release, etc.
7. For RNA or protein extraction, centrifuge the platelet sample at 800 x g for 10 min to pellet the platelets. Discard the supernatant completely without disturbing the pellet. Add the desired volume of RNA or protein lysis buffer to lyse the platelets.

2. Platelet Activation

1. In a tube, add 1 to 2 million purified platelets in up to 100 µL of staining buffer (PBS with 2% fetal bovine serum, FBS).
2. For a multiple number of samples, make a master mix of antibodies with 1 µL (0.5 mg/mL) of the following: PE-Cy7 rat anti-mouse TER 119, PE rat anti-mouse CD45, FITC rat anti-mouse CD41, and APC rat anti-mouse/human CD62P (P-selectin) to detect RBC, WBC, platelets, and activated platelets, respectively. Add the master mix to the tubes for staining the cells. Do not add thrombin.
3. In another tube, add 1 to 2 million purified platelets in up to 100 µL of staining buffer, and 0.4 mM GPRP peptide. Add thrombin to activate the platelets and stain the cells following step 2.2.
   NOTE: GPRP should be added to the sample before adding thrombin to prevent aggregate or clot formation through platelet activation. Thrombin concentration should be optimized to obtain good activation of platelets. After platelet activation, event counts are decreased during the acquisition of samples by flow cytometry.
4. As a positive control, add 1 µL of whole blood in up to 100 µL staining buffer in a tube and 0.4 mM GPRP peptide. Add thrombin to activate platelets. As a negative control, add 1 µL of whole blood in up to 100 µL of staining buffer. Do not add thrombin to the negative control sample. Stain the cells following step 2.2.
5. For flow cytometry isotype control, label 5 tubes with unstained, PE-Cy7, PE, FITC, and APC. Add 100 µL of staining buffer, 1 µL of blood, and 1 µL of the respective antibody to the labeled tubes to stain the cells.
6. Incubate samples to stain for 30 min at room temperature in the dark.
7. Wash the samples by adding 1 mL of staining buffer to each tube. Centrifuge at 800 x g for 5 min at room temperature. Discard the staining buffer carefully without dislodging the pellet.
8. Add 300 µL of staining buffer to each tube, mix mildly, and transfer to a FACS tube. Store the stained samples in the dark until analyzed with a flow cytometer.

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 the layers and right panel shows the actual image.

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 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