Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.
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. We describe here purification of platelets from mouse blood 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 recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.
Platelets are a component of blood that functions as an initiator of blood clotting in response to damage in the blood vessel. They gather at the site of injury to plug the vessel wall1. Platelets are anucleate fragments of cytoplasm derived from the megakaryocytes of the bone marrow under the influence of thrombopoietin and enter the circulation2. They are considered as metabolically active and capable of sensing extracellular environment by activating intracellular signaling cascades that result in platelet spreading, aggregation, and hemostatic plug formation1,3. Besides hemostasis/thrombosis and wound healing4, platelets play an important role in host inflammatory responses, angiogenesis, and metastatis3,5,6,7,8.
Platelets are purified from blood to study their biochemical and physiological properties, which should be free from other blood components. Since red blood cells (RBC) and white blood cells (WBC) contain significantly more RNA and proteins than platelets9,10, the presence of even a small number of these cells can interfere with transcriptomic and proteomic analyses of RNA and proteins derived from platelets. We found that purified platelets activated with thrombin bind antibody such as anti-GPIIb/IIIa (JON/A) and anti-P-selectin more efficiently than whole blood platelets.
Since platelets are fragile, it is important to treat the samples as mildly as possible. If the platelets are activated, they release their granule contents and ultimately degrade. Therefore, to keep the platelets’ functional properties intact, it is important to maintain platelet quiescence during isolation. Several protocols have described isolation of platelets from human, dog, rat, and non-human primate by various methods1,10,11,12. 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.
Ford and his colleagues described platelet purification from human blood using iohexol medium11. This method uses a similar volume of blood sample and medium during purification. Since humans yield a higher volume of blood, it is relatively easy to purify the platelets.
Iohexol is a universal density gradient inert medium that is freely soluble in water and used in the fractionation of nucleic acids, proteins, polysaccharides, and nucleoproteins13,14. It has low osmolality and is non-toxic, thus making it an ideal medium for purification of intact living cells11. It is a non-particulate medium; therefore, the distribution of cells in a gradient can be determined using hemocytometer, flow cytometer, or spectrophotometer. It does not interfere with most of the enzymatic or chemical reaction of the cells or cellular fragments after dilution.
The mouse serves as an important animal model for many human diseases15,16,17,18. There are a few published articles that describe purification of mouse platelets19,20. 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 is used, the platelet layer cannot be clearly separated from RBC-WBC layer after centrifugation. In this article, we have described a quick and simple method of mouse platelet purification with three-fold more iohexol gradient medium relative to the blood sample volume and low speed centrifugation. We have also activated the purified platelets with thrombin and investigated their quality with flow cytometry and microscopy.
Mouse blood collection should be conducted with appropriate institutional animal care and use committee approval.
NOTE: The platelet purification protocol is described in a flow diagram in Figure 1.
1. Collection of Blood
2. Platelet Purification
NOTE: Platelet purification should be carried out at room temperature to prevent their degradation. Make sure that temperature of the reagents and instruments are between 18 to 22 °C. To prevent platelet degradation, fast pipetting and vigorous shaking should be avoided during the procedure.
3. Counting the Platelets
4. Platelet Activation
5. Flow Cytometry Analyses of Platelets
The summary of platelet purification is described in a flow diagram (Figure 1). Steps include collection of blood from mouse using retro-orbital bleeding in the presence of an anticoagulant, addition of blood sample onto the iohexol gradient medium, centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The quality of the purified platelets was evaluated with microscopy and flow cytometry after staining with antibody to detect any contaminating cells and activated platelets.
The iohexol gradient medium and the blood sample formed two separate layers in the tube if the blood was slowly added onto the medium (Figure 2A). However, if there is delay in this step, most of the blood sample could diffuse into the medium and it might be difficult to purify the platelets. The wide-bore pipette tips ensured no physical stress to the platelets which is important for maintaining integrity of the platelets and preventing their degradation. During centrifugation, slow acceleration helped prevent inadvertent mixing of the blood sample with iohexol medium and slow deceleration helped maintain the gradient after centrifugation. The top (straw color) layer contained the plasma and did not contain any types of intact blood cells, the second layer (whitish) from the top contained majority of the platelets which was collected using a wide-bore pipette tip, the third layer (transparent) was platelet poor which was partly collected carefully to avoid aspiration of bottom layer (red) RBC and WBC (Figure 2B). The platelet-rich layer and platelet poor layer could not be seen separated if the platelet counts were low in the blood sample. The RBC and WBC layer could not be seen as separate layers since blood volume was small. However, WBC forms a white layer, called buffy coat between platelet poor layer and RBC layer if a larger volume of blood is used for purification10,11. It is important to carry out the whole procedure at 18 to 22 °C. Both the higher temperature and refrigeration of the samples yielded lower platelet counts due to degradation.
We found 18.2 to 38.5% (27.1 ± 6.5%, n=12) recovery/yield of the purified platelets. To investigate their viability, purified platelet samples were activated with thrombin. Flow cytometric analysis of platelets samples were done after staining with the markers for platelets, activated platelets, RBC, and WBC. The whole blood showed distinct populations of RBC, WBC, and platelets on a logarithmic forward scatter vs side scatter dot plot (Figure 3A), whereas the purified platelets showed a distinct population with negligible numbers of RBC and WBC (Figure 3B). The whole blood showed RBC and WBC after staining them with anti-mouse Ter119 and anti-mouse CD45 antibody (Figure 3C) whereas the purified platelets showed negligible number of RBC and WBC (Figure 3D). We evaluated the purified platelet sample with a microscope and did not see any significant number of intact RBC and WBC. Therefore, the RBC and WBC events which were shown in Figure 3D might be the fragments of RBC and WBC containing Ter119 and CD45, respectively. Purified platelets which were not treated with thrombin showed almost no activation indicating their resting state (Figure 3E), whereas purified platelets treated with thrombin showed 71% activation (up to 93% activation was observed in some samples) (Figure 3F) indicating their viability. We also performed microscopic evaluation of purified platelet samples not treated with thrombin which showed no platelet aggregation (Figure 4A), however, purified platelets formed aggregates after being treated with thrombin (Figure 4B), which further indicate their viability. Based on flow cytometric and microscopic studies, we confirmed that our purified platelets were good quality.
Figure 1: Schematic diagram for mouse platelet purification. Blood sample from mouse is collected in the presence of an anticoagulant. The blood sample is slowly added onto the Iohexol gradient medium and centrifuged at 400 x g for 20 min at 20 °C. The platelet layer is transferred to a new tube. The quality of purified platelets is checked with microscopy and flow cytometry. The platelets can be used immediately or stored at -80 °C. Please click here to view a larger version of this figure.
Figure 2:. 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. Please click here to view a larger version of this figure.
Figure 3: Flow cytometric analysis of purified platelets. (A) Whole blood shows RBC, WBC, and platelet populations. (B) Purified platelets show minor numbers of RBC and WBC events. (C) Whole blood shows RBC and WBC after staining with anti-mouse Ter119 and anti-mouse CD45. (D) Purified platelets show negligible number of RBC and WBC events after staining with anti-mouse Ter119 and anti-mouse CD45. (E) Purified platelets stained with anti-mouse CD41 and anti-mouse/human P-selectin show almost no platelet activation. (F) Purified platelets treated with thrombin and stained with anti-mouse CD41 and anti-mouse/human P-selectin show activation of platelets. Please click here to view a larger version of this figure.
Figure 4: Microscopic evaluation of purified platelets. (A) Purified platelets do not show any aggregation. (B) Purified platelets treated with thrombin show aggregation. Both figures are 200x magnification, ocular lens 10x and objective lens 20x. Please click here to view a larger version of this figure.
Commonly, platelets are isolated by low-speed centrifugation which yields platelet-rich plasma that contains a significant number of blood cells, cellular debris, and plasma proteins which can interfere with the biochemical and physiological assays and needs further purification21. Therefore, it is important to use a rapid and simple method which can yield pure platelets without major contaminants. The protocol presented here describes the purification of platelets from mouse blood using a gradient medium with low speed centrifugation. The parameters used in this protocol maximize the yield and minimize the degradation of platelets. The gradient medium and plasma proteins can be removed by including a washing step after platelet collection which can increase the sensitivity of platelets to agonist or antibody. Most importantly, the purified platelets are in resting state, but they can be activated in the presence of an agonist, such as thrombin. We have found that thrombin activity varies from one source to another. Therefore, thrombin concentration should be optimized empirically to obtain good activation of platelets.
Due to a small volume of mouse blood, it is inconvenient to purify platelets because platelets can be mixed with RBC and WBC during aspiration. We have resolved this issue by optimizing the ratio of blood sample and gradient medium. We have found that three-fold more gradient medium relative to the blood volume can clearly separate the platelet layer from the serum and RBC-WBC layers which facilitates easy collection of pure platelets.
Platelet purification should be carried out at room temperature between 18 to 22 °C to prevent their degradation. It has been reported that platelets degrade if stored in the refrigerator or temperatures above 27 °C11. Fast pipetting and vigorous shaking should be avoided to prevent platelet degradation during purification. Since different centrifuges have different programs, the acceleration/deceleration should be determined empirically to maintain the gradient.
If several mouse blood samples are used in the purification, the bleeding should be done in less than 1 h and purification should be completed next within 1 h. If blood samples are left for more than 2 h, platelets cannot be purified due to their degradation. If more platelets are needed for any study, the mouse blood can be collected by terminal procedures such as cardiac puncture or inferior vena cava bleeding, which yield 800 to 1,000 µL blood, and platelet purification should be carried out in multiple tubes and combined after purification.
In conclusion, we have described a simple and rapid protocol for platelet purification from a small volume of mouse blood. This method can also be used for platelet purification from other species as well. The purified platelets can be used for gene expression, activation and granule release, aggregation, and adhesion assays upon stimulation with various agonists.
The authors have nothing to disclose.
This work was supported by a start-up funding of Cincinnati Children’s Research Foundation and a University of Cincinnati Pilot Translational grant to M.N. We would like to thank the Cincinnati Children’s Hospital Research Flow Cytometry Core for their services.
APC rat anti-mouse/human CD62P (P-selectin) | Thermoscientific | 17-0626-82 | Platelets activation marker |
Eppendorf tube | Fisher Scientific | 14-222-166 | Tube for centifuge |
FACS DIVA software | BD Biosciences | Non-catalog item | Analysis of platelets and whole blood |
FACS tube | Fisher Scientific | 352008 | Tubes for flow cytomtery |
Fetal bovine serum (FBS) | Invitrogen | 26140079 | Ingredient for staining buffer |
FITC rat anti-mouse CD41 | BD Biosciences | 553848 | Platelets marker |
Flow cytometer | BD Biosciences | Non-catalog item | Analysis of platelets and whole blood |
FlowJo software | FlowJo, Inc. | Non-catalog item | Analysis of platelets and whole blood |
Gly-Pro-Arg-Pro (GPRP) | EMD Millipore | 03-34-0001 | Prevent platelet clot formation |
Hematocrit Capillary tube | Fisher Scientific | 22-362566 | Blood collection capillary tube |
Hemavet | Drew Scientific | Non-catalog item | Blood cell analyzer |
Hemocytometer | Hausser Scientific | 3100 | Cell counting chamber |
Isoflurane | Baxter | 1001936040 | Use to Anesthetize mouse |
Microscope (Olympus CKX41) | Olympus | Non-catalog item | Cell monitoring and counting |
Nycodenz (Histodenz) | Sigma-Aldrich | D2158 | Gradient medium |
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 |
Pipet tips 1000 µL, wide-bore | ThermoFisher Scientific | 21-236-2C | Transferring blood and platelet samples |
Phosphate buffured saline (PBS) | ThermoFisher Scientific | 14040-117 | Buffer for washing and dilution |
Sodium chloride | Sigma-Aldrich | S7653 | Physiological saline |
Sodium citrate | Fisher Scientific | 02-688-26 | Anti-coagulant |
Staining buffer | In-house | Non-catalog item | Wash and dilution buffer |
Steile water | In-house | Non-catalog item | Solvent |
Table top centrifuge | ThermoFisher Scientific | 75253839/433607 | Swinging bucket rotor centrifuge |
Thrombin | Enzyme Research Laboratoty | HT 1002a | Platelet activation agonist |
Tricine | Sigma-Aldrich | T0377 | Buffer for Nycodenz medium |