Platelets are one of the formed elements in the blood of mammals. They are small, biologically active cytoplasmic fragments shed from the cytoplasm of mature megakaryocytes in the bone marrow. Under normal physiological conditions, platelets circulate in a dispersed state within blood vessels. When blood vessels are damaged, blood rheology changes, or platelets are stimulated by chemical substances, they undergo a series of changes including deformation, adhesion, aggregation, and release, followed by cross-linking, which promotes thrombosis.
Platelets play a key role in the process of thrombosis. Preparing functional platelets or washed platelets serves as the experimental basis for investigating the effects and mechanisms of antithrombotic drugs on platelet function.
Platelet-Rich Plasma (PRP) Preparation Solution, also known as Platelet-Rich Plasma Isolation Solution, is designed to prepare functional platelets. It can separate platelets from other blood cell components while retaining them in the plasma, with simple and convenient operation. The reagent is not sterilized by filtration; 100 ml of this solution can be used to process 900 ml of fresh blood. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. Fresh whole blood
2. Micropipette, centrifuge tubes or test tubes, centrifuge, hemocytometer (optional), microscope (optional)
Operating Procedures (For Reference Only)
1. Collect fresh blood from donors who have not taken platelet function inhibitors (e.g., aspirin) within 2 weeks. Promptly mix the fresh blood with PRP Preparation Solution at a ratio of 9:1 in a centrifuge tube or test tube.
2. Centrifuge the mixture at 1000 rpm for 10 minutes. If the separation effect is unsatisfactory, extend the centrifugation time to 15 minutes.
3. Collect the supernatant, which is the platelet-rich plasma. Avoid aspirating the middle layer during collection.
4. For platelet counting (if needed), dilute the PRP with platelet diluent. Then place the diluted sample on a hemocytometer and count the platelets in five medium-sized squares (the four corner squares and the central square) under the high-power lens of a microscope.
5. Calculation formula: Platelet count (per L) = Number of platelets in 5 medium-sized squares × 10⁹/L
6. Store the prepared PRP at 22°C (room temperature) or 4°C generally. For long-term storage, add 6% DMSO. Note that platelets maintain their biological activity for a short period in vitro; it is recommended to complete subsequent downstream experiments within 6 hours.
Result Analysis
The prepared platelet-rich plasma achieves the separation of platelets from other blood cell components, while keeping platelets combined with plasma rather than isolating them.
Precautions
1. When collecting blood, the needle insertion depth should be slightly deeper. Wipe off the first drop of blood, then collect the blood sample for platelet counting immediately. Perform all operations rapidly to prevent platelet aggregation.
2. This reagent separates platelets from other blood cell components without separating them from plasma, featuring simple and feasible operation.
3. After loading the platelet suspension into the counting chamber, allow it to stand undisturbed for 10-15 minutes. If the room temperature is too high, maintain humidity around the counting chamber to prevent water evaporation. Use moderate light intensity during counting, and distinguish between refractive platelets and impurities. Counting under a phase-contrast microscope yields better and more accurate results.
4. Use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
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