Purification and Activation of Platelets from Murine Whole Blood

To purify the platelets, use a wide-bore pipette tip to layer 200 microliters of freshly-harvested whole blood, slowly down the side of a 1.5-milliliter tube onto 600 microliters of iohexol gradient medium without mixing.

After centrifugation in a swinging bucket rotor to isolate the platelets, use a new wide-bore pipette tip to collect most of the platelet-rich layer, and a small fraction of the platelet-poor layer without aspirating the white blood cell or red blood cell layer. Add the platelet sample to a new tube, and add 1 milliliter of PBS to the platelets.

Mix the platelets by inversion, before performing another centrifugation. Then, resuspend the pellet in 200 microliters of PBS with mild pipetting. For platelet activation, transfer 1 to 2 times 10 to the 6th platelets to a new tube containing 100 microliters of staining buffer. Add 1 to 2 times 10 to the 6th platelets to a different tube containing 100 microliters of staining buffer supplemented with 0.4 millimolar GPRP peptide.

Add thrombin to the tube with the GPRP peptide to activate the platelets, and add the antibody cocktail of interest to both tubes. As a positive control, add 1 microliter of whole blood in up to 100 microliters of staining buffer in a tube containing 0.4 millimolar GPRP peptide, and add thrombin to activate these platelets.