Purification Platelets from Mouse Blood with Sickle Cell Disease Using Iohexol Gradient Medium

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. Since RBC and WBC contain significantly more RNA and proteins than platelets, the presence of even a small number of these cells can interfere with transcriptomic and proteomic analyses of RNA and proteins derived from platelets. Several protocols have described the isolation of platelets from human, dog, rat, and non-human primate by various methods. Some of the methods require multiple steps such as collection of platelet-rich plasma by centrifugation, filtration by separation column, negative selection of platelets with RBC- and WBC-specific antibody conjugated to magnetic beads, and so on, which are time-consuming and may degrade platelets and their contents. Moreover, mouse blood with sickle cell disease contains a significant level of fragments of RBC which should be removed from the platelet preparation.

However, the mouse yields a relatively smaller volume of blood, which makes it difficult to purify platelets. If the same small volume of gradient medium and blood samples are used, the platelet layer cannot be clearly separated from RBC-WBC layer after centrifugation. We describe a simple method for purification of platelets from mouse blood with sickle cell disease using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The platelet layer is collected and centrifuged again at 200 x g for 20 min at 20 °C to remove the residual fragments of RBC. The recovery/yield of the purified platelets were 10-17%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets were activated with thrombin indicating their viability. We confirmed that the purified platelets were sufficiently pure using flow cytometric and microscopic evaluation. Although flow cytometric analysis of purified platelet from sickle cell disease mice showed a few RBC events after staining with anti-TER119 antibody, the microscopic study did not show any intact RBC or larger fragments indicating that these are smaller fragments of RBC which do not interfere with the biochemical and functional studies. This method can be used for purification of platelets from the blood of other species and disease models as well.