Separation of Platelets from Whole Blood

Purpose

This protocol describes how to isolate human platelets from whole blood. Isolated platelets are used for static adhesion assays, for flow chamber assays, flow cytometry measurements, etc.

Materials

• Citrate buffer (25 g sodim citrate, 8 g citric acid, 500 ml H₂ O₂ ).
• Prostaglandin E₁ (PGE₁ ).
• ACD buffer (6.25 g sodium citrate.2 H₂ O , 3.1 g citric acid anhidrous, 3.4 g D-glucose in 250 ml H₂ O).
• Hepes-Tyrode buffer pH 7.4 (134 mM sodium chloride, 12 mM sodium bicarbonate, 2.9 mM potasium chloride, 0.34 mM sodium phosphate monobasic, 1 mM magnesium chloride (avoid for adhesion assay), 5 mM Hepes, 5 mM glucose, 1 % BSA).

Procedure

1. Draw 45 ml blood into a syringe containing 5 ml citratebuffer. Optional: Add 2 micromolar PGE1 to avoid platelet activation.
2. Transfer to a 50 ml tube and centrifuge at 150g for 15 minutes at room temperature.
3. Carefully transfer the upper phase (Platelet Rich Plasma) to a 15 ml tube and add 1/10 volume of ACD anticoagulant.
4. Pellet platelets by centrifugation at 900 g for 5 minutes at room temperature. Note: After centrifugation, supernatant still contains a significant number of platelets and can be collected for experiments.
5. Resuspend the pellet in 5-10 ml Hepes-Tyrode buffer.

Keep platelets at room temperature throughout the experiment. Freshly isolated platelets should be used within 2 hours.