Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

Our research uses multicolor flow cytometry to identify and characterize platelet subpopulations, aiming to understand the roles, activation patterns, and clinical relevance in thrombosis, inflammation and disease specific context. The established multicolor flow cytometric assay enabled to observe the , a platelet agonist induces a distinct activation pattern and compared to collagen related peptide generates a procoagulant and of subpopulation. The multicolor assay enables the simultaneous measurement of platelet activation, adhesion, aggregation, and apoptotic potential of a single platelet within a population of isolated human platelets in a single tube.

[Instructor] To begin, prepare all the reagents required for the procedure. Now, mix 10 milliliters of 10x Tyrode's buffer with 90 milliliters of distilled water. Add 0.1 gram of glucose and adjust the pH of the full volume with hepes to reach 7.4. First, set the pH of 10 milliliters of the buffer to 7.4, and then adjust the pH of the remaining 90 milliliters with one normal hydrochloric acid to 6.5. For blood sample collection, prepare a 10 milliliter syringe for each donor with two milliliters of ACD anticoagulant. Let the syringe equilibrate at room temperature. After collecting donor blood in a prewarmed ACD syringe, slowly transfer the ACD treated] blood into a 15 milliliter reaction tube. Centrifuge the ACD treated blood at 209G for 20 minutes at room temperature without using the break. Meanwhile, prepare 25 milliliters of Tyrode's Hepes buffer, pH 6.5 in a 50 milliliter reaction tube. After centrifugation, gently transfer the upper platelet rich plasma layer into the tube containing Tyrode's Hepes buffer. To prevent contamination, leave approximately one milliliter of platelet rich plasma above the buffy coat and erythrocyte layer. Then, centrifuge the suspension at 430G for 10 minutes at room temperature to pellet the platelets. Discard the supernatant and carefully resuspend the platelet pellet in 200 to 300 microliters of Tyrode's buffer, adjusted to pH 7.4. Measure the platelet count using a cell counter before adjusting it to 200,000 platelets per microliter with Tyrode's Hepes buffer at pH 7.4. For receptors that do not require activation prior to staining, including CD61, CD42B, CD41, CXCR7, and CXCR4, prepare one unstained and one stained sample per antibody. For receptors or markers that require activation such as PAC-1, CD63, CD62P, NXN5, and zombie NIR. Activate the sample with 10 micrograms per milliliter of CRPXL and prepare both activated and nonactivated samples. To activate the platelets, incubate them with 10 micrograms per milliliter of CRPXL for 30 minutes. Then, incubate the isolated platelet with varying concentrations of the fluorochrome conjugated antibody or die for 30 minutes. Then, add 300 microliters of anexin binding buffer to each sample to dilute it. Measure 10,000 events for each sample using a flow cytometer. Calculate the stain index for each antibody to determine the optimal separation between positive and negative populations. Plot the stain index against antibody concentrations to identify the optimal concentration for each antibody For staining, dissolve hemen powder in 1.4M Sodium Hydroxide to create a 3M solution. Heat the solution to 96 degrees Celsius for five minutes at 450 revolutions per minute. Dilute it with distilled water to obtain a three millimolar stalk solution. After adjusting the platelet count to 1 million platelets per sample, activate the platelets as demonstrated earlier. Add 43.3 microliters of antibody cocktail to each sample and incubate them for 30 minutes at room temperature in the dark. After incubation, dilute each sample with 300 microliters of one X and X in binding buffer composed of 10 millimolar Hepes Sodium Hydroxide at pH 7.4. Measure each stained sample immediately using the flow cytometer. For compensation with beads, prepare 150 microliters of PBS for each fluorophore and add one drop of beads and one microliter of the fluorescence conjugated antibody. To compensate for annexin V, use PECY7B seven beads labeled with anti-human CD 62L conjugated to the same fluorophore as annexin V. Use amine reactive compensation beads for the Amine reactive dye by mixing two drops of positive beads and one drop of negative beads in 150 microliters of PBS with one microliter of die. Confirm that the positive beads react and display a signal and the negative beads do not. Prepare a fluorescence minus one control for every marker. The scatter plots showed a clear shift in the platelet population. With hemin forming more platelets with low forward scatter than other agonists. Treatment with ADP thrombin and CRPXL resulted in 40 to 60% platelets being CD42 be positive, while hemin significantly reduced this population. All platelet agonists had no effect on CD41 and CD61 surface expression. Only hemen significantly increased the CXCR4 and CXCR7 surface expression. In addition, the phosphatidylserine exposure was also enhanced under hemin treatment, resulting in an enhancement of annexin V positive cells. UMAP and phenol graph analyses defined 27 distinct platelet clusters, which varied with agonist type. Hemin caused the most dramatic shift in cluster patterns. Resting platelet subpopulations were most preserved with ADP and leased with hemin.