Name: Analyzing Platelet Subpopulations by Multi-color Flow Cytometry (Video) | JoVE Journal
Uploaded: 2025-06-10T13:00:00.000+00:00
Duration: 8 min 4 s
Description: Learn multi-color flow cytometry to identify platelet subpopulations and analyze activation patterns with various agonists. This method reveals distinct clusters for studying thrombosis and inflammation.

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5 minutes

The project was supported by the German Research Foundation (DFG) - Project number 335549539 - GRK 2381.

Blood collection and handling for research purposes were approved by the Ethics Committee at the Medical Faculty of the Eberhard Karls University and the University Hospital of T&#252;bingen (ethics vote 238/2018B02), and the study methodologies conformed to the standards set by the Declaration of Helsinki. Healthy donors gave written informed consent for blood collection. The baseline characteristics for healthy donors are listed in Table 1. Figure 1 gives an overview of the flow cytometry protocol.
1. Isolation of human platelets from whole blood
NOTE: ISTH guidelines were followed in terms of sample collection, tourniquets, and the choice of anticoagulant in order to ensure a standardized manner to isolate platelets20.
<ol>
	<li>Buffer preparation
	<ol>
		<li>Acid-citrate-dextrose (ACD) anticoagulant buffer: Dissolve 12.50 g Na3-Citrat (85 mM C6H5Na3O7 x 2 H2O), 6.82 g citrate acid (71 mM) and 10.00 g glucose (111 mM) in 475 mL of aqua destillata. Adjust the pH with 1 M NaOH to 4.69. Fill up to 500 mL with aqua destillata. Then, filter sterilize the buffer and store at 4 &#176;C until use. 
		NOTE: All solutions are filter sterilized with a vacuum-driven filter system, which uses a 0.22 &#181;m pore size fast flow polyether sulfone membrane.</li>
		<li>Tyrode&#39;s buffer (10x): Weigh 80.00 g NaCl, 10.15 g NaHCO3, and 1.95 g KCl and add aqua destillata to 1 L. Filter sterilize the buffer and store at 4 &#176;C until use.</li>
		<li>Tyrode&#39;s-HEPES buffer (1x; 137 mM NaCl, 2.8 mM KCl, 12 mM NaHCO3, 5 mM glucose, 10 mM HEPES): Mix 10 mL of 10x Tyrode&#39;s buffer and 90 mL of aqua destillata. Dissolve 0.1 g glucose and 0.1 g bovine serum albumin (BSA). Adjust the pH with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) to 7.4. Set 10 mL of Tyrode&#39;s buffer to pH 7.4 and adjust the pH of the residual 90 mL of Tyrode&#39;s to 7.4 with 1 N HCl to pH 6.5. 
		NOTE: BSA could scavenge some chemicals, such as hemin. In this case, it&#39;s important to prepare the 1x Tyrode&#39;s buffer without BSA. All buffers must be allowed to warm up to room temperature or 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Blood sample collection
	<ol>
		<li>Prepare for every donor one 10 mL syringe with 2 mL of ACD anticoagulant and let it adjust to room temperature or 37 &#176;C using an incubator. Keep the dilution factor of 1:5 (ACD: blood). 
		NOTE: Use smaller or larger syringes to draw blood samples. Cold ACD buffer could pre-activate the platelets and impair your results.</li>
		<li>Use a butterfly needle (21G) to slowly collect the donor blood in the pre-warmed ACD syringes. Open the tourniquet hose before filling the syringe to avoid pre-activation. 
		NOTE: Syringes with a larger gauge needle can minimize the shear-induced activation of platelets.</li>
		<li>Slowly transfer the ACD blood into a 15 mL reaction tube. Use the blood immediately to avoid pre-activation of the platelets.</li>
	</ol>
	</li>
	<li>Platelet isolation
	<ol>
		<li>Centrifuge the ACD blood for 20 min at 209 x g (room temperature, RT) without brakes. Prepare 25 mL of Tyrode&#39;s-HEPES buffer pH 6.5 in a 50 mL reaction tube.</li>
		<li>Gently transfer the resulting platelet-rich plasma (PRP, upper layer) into the 50 mL reaction tube containing Tyrode&#39;s-HEPES buffer, pH 6.5.</li>
		<li>Leave approximately 1 mL of PRP above the buffy coat and erythrocytes to avoid contamination with other blood cells.</li>
		<li>Centrifuge the suspension at 430 x g for 10 min at RT to pellet the platelets. Carefully resuspend the resulting pellet in 200-300 &#181;L of Tyrode&#39;s buffer, pH 7.4.</li>
		<li>Use a cell counter to determine the platelet count. Adjust the platelet count with Tyrode&#39;s-HEPES-buffer pH 7.4 to 200 x 103 platelets/&#181;L. See step 3 for further processing of platelet staining.</li>
	</ol>
	</li>
</ol>
2. Selection and titration of antibodies for flow cytometry
<ol>
	<li>Selection of antibodies and dyes
	<ol>
		<li>Select the antibodies and dyes with suitable fluorophores for the flow cytometer21. Note the filter configuration recommended by the flow cytometer manufacturer. An example of platelet markers and fluorophore configuration is given in Table 2 and Figure 2 for the following cytometer: BD LSR Fortessa with Laser configuration: violet (402 nm), blue (488 nm), yellow/green (561 nm), red (640 nm). 
		NOTE: Use brighter fluorochromes for lower-expressed antigens. For example, the bright fluorochromes BV412 and BV650 were used for the weakly expressed chemokine receptors (ACKR3 and CXCR4)22,23,24.</li>
	</ol>
	</li>
	<li>Titration of antibodies and dyes
	<ol>
		<li>Titrate the antibodies to find the right working concentration. For titration, use isolated human platelets from step 1 and a concentration series of the antibody. 
		NOTE: The working concentrations given by the manufacturer are generally a good starting point for the concentration series.
		<ol>
			<li>If working with antigens that only get expressed under treatment, activate the platelets prior to staining to be sure that negative and positive cells can be detected as well. For example, PAC-1 binds only to activated integrin &#945;IIb&#946;3. Therefore, strongly activate the platelets to get a bright, positive signal, e.g., with 10 &#181;g/mL of collagen-related peptide (CRP; see Figure 3).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Titration of samples
	<ol>
		<li>Isolate the human platelets as in step 1 and adjust the platelet count to 1 x 106 platelets per sample.
		<ol>
			<li>For receptors that need no activation prior to staining: CD61, CD42b, CD41, CXCR7, CXCR4 use one unstained and a stained sample per antibody. For receptors/markers that need to be activated with, for example, 10 &#181;g/mL of CRP-XL prior to staining: PAC1, CD63, CD62P, Annexin V, Zombie NIR, use one activated and one non-activated sample15,25,26.</li>
		</ol>
		</li>
		<li>Activate the platelets (1 x 106 platelets per sample) if necessary for 30 min with 10 &#181;g/mL CRP-XL. Then, incubate the isolated platelets (1 x 106 platelets per sample) for 30 min with different concentrations of the fluorochrome-conjugated antibody or dye and add 300 &#181;L of 1x&#160;Annexin Binding Buffer to dilute the sample (examples for concentrations are given in Table 3).</li>
		<li>Measure 10,000 events for every sample.</li>
		<li>Calculate the stain index that shows the optimal separation of the positive and negative populations for every antibody, as shown in Supplementary Figure 1.</li>
		<li>Plot the stain index for each concentration of antibody to find the optimal concentration.</li>
	</ol>
	</li>
</ol>
3. Staining for flow cytometry
NOTE: See Table 4 for a staining example with four different platelet activators (CRP-XL, ADP, thrombin, hemin).
<ol>
	<li>Hemin needs to be prepared freshly. Dissolve the powder in 1.4 M NaOH to a 3 M solution and heat it to 96 &#176;C for 5 min at 450 rpm. Then, dilute it with distilled H2O to a 3 mM stock solution.</li>
	<li>After adjustment of the platelet count (per sample: 1 x 106 platelets), activate the platelets in a tube with the platelet activators for 30 min at RT as given in Table 4.</li>
	<li>Add 43.3 &#181;L of antibody cocktail to the samples and incubate the platelets for 30 min at RT in the dark.</li>
	<li>After incubation, dilute the samples with 300 &#181;L of 1x Annexin Binding Buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) so that the Annexin V protein can bind to the phosphatidylserine on the outer side of the cell membrane. Measure the samples immediately. 
	NOTE: Measure the samples immediately after preparation. As the samples are not fixed, delaying sample preparation and measurement can increase the variability of the results. Fixation impairs Annexin V binding.</li>
</ol>
4. Compensation setup and fluorescence minus one control (FMO) measurement
NOTE: Before recording data, run a compensation setup. A flow cytometer automatically calculates compensation settings; refer to the usual manuals of the flow cytometer for the particular compensation procedure. Nevertheless, use compensation controls either with cells or with beads.
<ol>
	<li>Compensation setup
	<ol>
		<li>For compensation with beads, prepare 150 &#181;L of phosphate-buffered saline (PBS) for each fluorophore with one drop of beads and 1 &#181;L of the fluorescence-conjugated antibody.</li>
		<li>Monitor the species&#39; reactivity of the beads. Use anti-mouse IgG&#954;&#160;and the negative control of polystyrene microparticles. To compensate for Annexin V, label PE/Cy7 beads with antibody PE/Cy7 anti-human CD62L that conjugates the same fluorophore as Annexin V.</li>
		<li>Use amine-reactive compensation beads for the amine-reactive dye. Use two drops of positive and one drop of negative beads in 150 &#181;L of PBS with 1 &#181;L of the dye. Check that the amine-reactive compensation beads react with the dye and show a positive signal. Check that the negative beads do not react with the amine-reactive dye and present a negative signal. 
		NOTE: For Zombie NIR APC-Cy7, use negative and positive amine reactive beads.</li>
		<li>Incubate the compensation samples for 30 min at room temperature in the dark.</li>
		<li>Run every compensation tube separately and gate on the singlet bead population based on FSC and SSC. The bead population appears as one small concentrated population. Draw a gate around this population.</li>
		<li>Display the histograms for every compensation tube and adjust the compensation values until the positive peaks of the different antibodies and dyes do not overlap. Then, measure every single compensation tube and let the spillover of the fluorophores be calculated as per the manufacturer&#39;s SOP.</li>
		<li>Proceed with acquiring the actual staining experiment with the isolated human platelets (see step 3 and Table 4). Adjust the SSC/FSC voltage before measurement to find the platelet population. Since CD61 is ubiquitously expressed on platelets, check with this fluorescence threshold if every size of the platelet can be detected.</li>
		<li>Then, measure 20,000 events per sample.</li>
	</ol>
	</li>
	<li>FMO measurement
	<ol>
		<li>Prepare for every marker an FMO control as per Table 5. In order to be able to determine the overlaps of all markers, prepare both unstimulated and stimulated samples.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Analysis with a flow cytometry data analysis software (Figure 4)
	<ol>
		<li>Drag and drop the FSC files into the software. Define with the FMO control the positive population for each marker.
		<ol>
			<li>Determine FMOs by plotting each FMO control against each marker in a dot plot. This makes the spill over into other channels visible. Use the FMO gate with the highest fluorescence spillover as the boundary to the positive population for the treated 10-color samples.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analyzing with a cloud-based flow cytometry analysis (Figure 5)
	<ol>
		<li>Open the software and create a new dataset. Drag and drop the FCS files acquired with the flow cytometer.</li>
		<li>Start the analysis by creating a workflow. The compensation matrix is automatically created from the raw data files.</li>
		<li>Run a UMAP analysis with the following settings: Neighbors: 15, Minimum Distance: 0.4; Components: 2; Metric: Euclidean; Learning Rate: 1; Epochs: 200; Embedding Initialization: spectral. Select all files and all 10 markers (features).</li>
		<li>Add Phenopraph analysis as a new task and run the analysis with the following settings: K Nearest Neighbors: 20; Distance Metric: Euclidean; Louvain Runs: 1; Number of Results: 1.</li>
		<li>Add a gating task to indicate the generated clusters. After running the analysis, it is possible to present the data in different blots, which can be exported.</li>
	</ol>
	</li>
</ol>

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<li>Hashemzadeh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+review+of+the+ten+main+platelet+receptors+involved+in+platelet+activity+and+cardiovascular+disease%5BTitle%5D)+AND+%22Am+J+Blood+Res%22%5BJournal%5D)">A comprehensive review of the ten main platelet receptors involved in platelet activity and cardiovascular disease.</a> Am J Blood Res. 13 (6), 168-188 (2023).</li>
<li>Moroi, M., Farndale, R. W., Jung, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation-induced+changes+in+platelet+surface+receptor+expression+and+the+contribution+of+the+large-platelet+subpopulation+to+activation%5BTitle%5D)+AND+%22Res+Pract+Thromb+Haemost%22%5BJournal%5D)">Activation-induced changes in platelet surface receptor expression and the contribution of the large-platelet subpopulation to activation.</a> Res Pract Thromb Haemost. 4 (2), 285-297 (2020).</li>
<li>Busuttil-Crellin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Guidelines+for+panel+design%2C+optimization%2C+and+performance+of+whole+blood+multi-color+flow+cytometry+of+platelet+surface+markers%5BTitle%5D)+AND+%22Platelets%22%5BJournal%5D)">Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers.</a> Platelets. 31 (7), 845-852 (2020).</li>
<li>Fink, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Subtilisin-Like+Protease+Furin+Regulates+Hemin-Dependent+Ectodomain+Shedding+of+Glycoprotein+VI%5BTitle%5D)+AND+%22Thromb+Haemost%22%5BJournal%5D)">The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.</a> Thromb Haemost. 123 (7), 679-691 (2023).</li>
</ol>

The authors have nothing to disclose.

Multi-color flow cytometry allows to define distinct platelet clusters/subpopulations associated with discrete functions12,17,18. Decisive for the results of the subpopulations and the receptor surface expression is the quality of the isolated human platelets. Therefore, one key step in the presented protocol is the preparation of the platelets. They need to be gently isolated to avoid activation prior to sample treatment with d...

Thrombocytes, also called platelets, are small, nucleus-free blood cells essential for hemostasis and the development of thrombosis1,2. They can be activated by various platelet agonists. Classical, physiological platelet agonists are adenosine diphosphate (ADP), which binds to the P2Y12 and P2Y1 receptors, thrombin that activates platelets through the PAR1 and PAR4 receptors, and collagen that interacts with glycoprotein VI. Collagen can be substituted in in vitro experiments by the collagen-related peptide (CRP-XL)3,4. Recently, hemin has been shown to induce platelet activation by the interaction of C-type lectin-like receptor (CLEC-2) and glycoprotein VI (GPVI), establishing hemin as a new endogenous platelet activator/agonist5,6. Several pathological situations can lead to hemolysis, resulting in the liberation of free ferric iron-containing hemin. All these platelet agonists alter the surface receptor expression on platelets. The alteration of the surface receptors and plasma membrane depends on the strength and concentration of the platelet agonist7,8,9. Therefore, it is important to phenotypically characterize platelets to be able to assign platelet subpopulations to platelet function.
High-dimensional methods such as flow cytometry have revealed that platelets form subpopulations with distinct functions. The subpopulations can be classified by different surface markers. In the literature, it is well known that two distinct platelet subpopulations are present: the procoagulant, which is characterized by phosphatidylserine externalization, and the aggregatory phenotype that activates integrin &#945;IIIb&#946;3. In addition to the classical activation markers, fluorescence dyes such as Glutathione-S-Aryloxide (GSAO) that detects mitochondrial function and oxidative stress can be used for further categorization, e.g., procoagulant platelets by their metabolic state10. This allows a distinction to be made between ballooning and mitochondrial permeability transition pore formation (MPTP) procoagulant platelet phenotype11.
Heemskerk et al. have shown by analyzing phosphatidylserine (PS) exposure and integrin &#945;IIb&#946;3 activity that different platelet populations can control distinct coagulation steps and that strong activators such as thrombin and collagen are required for phosphatidylserine exposure12. Further, activating the platelets by GPVI with collagen or the specific GPVI agonist convulxin is primarily responsible for the formation of adherent procoagulant platelets. Likewise, arachidonic acid, which leads to platelet activation by producing thromboxane A2 by cyclooxygenase, induces PS exposure13. However, arachidonic acid is, in comparison to thrombin or collagen, a weak platelet activator14. Van Velzen et al. also evaluated platelet surface antigens (CD42a/b, CD36, CD41, CD61) and activation markers (PAC-1, CD63, CD62P) in two separate measurements15. Additionally, van Velzen et al. were able to analyze blood from thrombasthenia patients and compared the subpopulations between healthy and diseased patients. Hindle et al. measured the influence of prostacyclin on platelet subpopulations after thrombin and CRP-XL treatment with two individual panels consisting of four markers each (PAC-1, Annexin V, CD62P, CD42b, or CDCD154, CD62P, CD63, CD42b)16. Recently, multi-color flow cytometry has been used to define resting (CD42b+, PAC1-, CD62P-, Annexin V-), aggregatory (CD42b+, PAC1+, CD62P+, Annexin V-) procoagulant (CD42b+, PAC1-, CD62P+, Annexin V+) and apoptotic (Cd42b-, PAC1-, CD62P-, Annexin V+) platelet subpopulations7,17. Laspa et al. extended/refined the multi-color flow cytometry approach by the addition of antibodies against the chemokine receptors CXCR4 and ACKR318. Based on the available literature, this is the first 10-color flow cytometer panel for isolated human platelets that allows the detection of 10 different antigens on an individual platelet (single-cell level) within a population of isolated human platelets in one tube. Hence, it is possible to simultaneously measure activation, adhesion, and aggregation potential, chemokine receptors, and apoptotic potential of a single cell to further distinguish the existing subpopulation. Thus, it complements Johnson et al., who defined the four main subpopulations with four markers (resting, procoagulant, aggregatory, apoptotic)17. So, establishing a multi-color flow cytometry assay enables the comparison of different platelet agonists in regard to their activation pattern and the formation of platelet subtypes within classical platelet subpopulations.
The method can be used for basic research regarding the analysis of platelet function and physiology. Moreover, the established multi-color flow cytometry assay for human platelets can be performed with platelets gained within patient studies, e.g., researching cardiovascular diseases and thrombotic events, to decode the platelet subpopulations induced by these diseases and the effects of treatments on these induced phenotypes. For example, Mueller et al. have shown that the platelet subpopulations can differ in aortic stenosis patients19. The gained knowledge might offer new openings for pharmaceutical interventions.

<table><tbody><tr><td>10x Binding Buffer&amp;nbsp;&amp;nbsp;</td><td>Invitrogen,&lt;br/&gt; Carlsbad, California, USA</td><td>88-8103-74</td><td>Buffer&amp;nbsp;</td></tr><tr><td>Adenosine diphosphate (ADP)</td><td>Probe &amp;amp; go Labordiagnostica GmbH&lt;br/&gt; Lemgo, Germany&amp;nbsp;&amp;nbsp;</td><td>70212</td><td>platelet agonist&amp;nbsp;</td></tr><tr><td>Annexin V PE-Cyanine7</td><td>Invitrogen,&lt;br/&gt; Carlsbad, California, USA</td><td>88-8103-74</td><td>dye&amp;nbsp;</td></tr><tr><td>anti-CD41 AF 700</td><td>BioLegend,&lt;br/&gt; San Diego, CA, USA</td><td>303728</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CD41 Alexa Fluor 700</td><td>BioLegend,&lt;br/&gt; San Diego, CA, USA</td><td>303727</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CD42b PerCP/Cyanine5.5&amp;nbsp;</td><td>BioLegend,&lt;br/&gt; San Diego, CA, USA</td><td>303918</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CD61 BV605</td><td>BD Bioscience&lt;br/&gt; Franklin Lakes, NJ, USA&amp;nbsp;</td><td>744382</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CD62P PE</td><td>Beckman Coulter,&lt;br/&gt; Brea, California, USA</td><td>IM1759U</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CD63 PE/Cy5&amp;nbsp;</td><td>Abcam,&lt;br/&gt; Cambrigde, UK</td><td>ab 234251</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CXCR4 BV650</td><td>BD Bioscience&lt;br/&gt; Franklin Lakes, NJ, USA</td><td>740599</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-CXCR7 BV421&amp;nbsp;</td><td>BD Bioscience&lt;br/&gt; Franklin Lakes, NJ, USA&amp;nbsp;</td><td>566234</td><td>antibody&amp;nbsp;</td></tr><tr><td>anti-PAC-1 FITC</td><td>BD Bioscience&lt;br/&gt; Franklin Lakes, NJ, USA</td><td>340507</td><td>antibody&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Applichem,&lt;br/&gt; Darmstadt, Germany</td><td>A1391</td><td>-</td></tr><tr><td>Citrit acid&amp;nbsp;</td><td>Carl Roth,&lt;br/&gt; Karlsruhe, Germany</td><td>6490.1</td><td>-</td></tr><tr><td>CRP-XL</td><td>CambCol Laboratories,&lt;br/&gt; Ely, UK</td><td>CRP-XL</td><td>platelet agonist&amp;nbsp;</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma Aldrich Co.,&lt;br/&gt; St. Louis, Missouri, USA</td><td>G7528</td><td>-</td></tr><tr><td>Dubecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich Co,&lt;br/&gt; St. Louis, Missouri, USA</td><td>D8537</td><td>-</td></tr><tr><td>Hemin&amp;nbsp;</td><td>Sigma Aldrich Co.,&lt;br/&gt; St. Louis, Missouri, USA</td><td>H9039-10G</td><td>platelet agonist&amp;nbsp;</td></tr><tr><td>HEPES</td><td>Carl Roth,&lt;br/&gt; Karlsruhe, Germany</td><td>9105.3</td><td>-</td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>Carl Roth,&lt;br/&gt; Karlsruhe, Germany</td><td>6781.3</td><td>-</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Carl Roth,&lt;br/&gt; Karlsruhe, Germany</td><td>3957.1</td><td>-</td></tr><tr><td>Sodium hydrogencarbonate&amp;nbsp;&amp;nbsp;</td><td>Sigma Aldrich Co,&lt;br/&gt; St. Louis, Missouri, USA</td><td>401676</td><td>-</td></tr><tr><td>Thrombin&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>10602400001</td><td>platelet agonist&amp;nbsp;</td></tr><tr><td>tri-Sodium citrate-dihydrat&amp;nbsp;</td><td>Applichem,&lt;br/&gt; Darmstadt, Germany</td><td>1416551211</td><td>-</td></tr><tr><td>Zombie NIR</td><td>BioLegend,&lt;br/&gt; San Diego, CA, USA</td><td>423105</td><td>amine-reactive dye&amp;nbsp;</td></tr></tbody></table>

analyzing platelet subpopulations by multi-color flow cytometry

Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can cause bleeding or thrombotic events in patients with cardiovascular diseases. Therefore, it is important to characterize platelets phenotypically to be able to assign platelet subpopulations to platelet function. Stimulation of human platelets with platelet agonists and activators induces morphological and physiological changes in platelets, accompanied by changes in the surface receptor population. This leads to functionally diverse platelet subpopulations. Classically defined platelet subpopulations are resting, aggregatory, procoagulant, and apoptotic platelets. To characterize the effect of agonists on platelet subtypes, we established an assay using multi-color flow cytometry consisting of 10 different antibodies and dyes (anti-CD62P, anti-CD63, anti-CD61, anti-CD41, anti-CD42b, anti-Integrin&#945;IIb&#946;3 (clone: PAC-1), anti-CXCR4, anti-ACKR3, Annexin V, Zombie NIR). Isolated human platelets were incubated with platelet agonists and stained with specific fluorophore-conjugated antibodies and dyes. Afterwards, they were measured by flow cytometry. This allows us to define agonist-specific subtypes within the classic four subpopulations. In conclusion, the combination of activation markers (anti-CD62P, anti-CD63, anti-Integrin&#945;IIb&#946;3 (clone: PAC-1)), inflammatory markers (anti-CXCR4, anti-ACKR3) and apoptotic markers (Annexin V, Zombie NIR) that compose the 10-color flow cytometry panel described in this manuscript opens up the possibility to define further platelet subtypes that could be linked to specific platelet function. This method can be applied in basic research on platelet function and physiology, as well as in defining new platelet subtypes in disease models and patient studies.

There are two options for analyzing the raw data collected by a multi-color flow cytometry panel. One option is to analyze it manually with the analysis software (Figure 3 and Figure 4). Therefore, the raw data needs to be dragged and dropped into the software window. First, the cells need to be found by adjusting the SSC/FSC scale in the scatterplot display mode to show the platelet population as a distinct point cloud. Then, the positive cells for each marker ...

Multi-color flow cytometry for platelets can identify new platelet subtypes within classical platelet subpopulations, such as resting, aggregatory, procoagulant, and apoptotic platelets. This allows comparison of different subpopulation patterns induced by various platelet agonists. Here, a procedure for establishing a multi-color flow cytometry panel is described in detail.

Platelets, or thrombocytes, are small anucleated blood cells that play a crucial role in hemostasis and thrombosis. Defects in platelet functions can ...

analyzing-platelet-subpopulations-by-multi-color-flow-cytometry

Research

JoVE Journal

Biochemistry

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

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.

Purification of Platelets from Mouse Blood

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

Presented here is a flow cytometry-based technique that allows for simultaneously measuring the number of cell divisions, surface cell phenotype, and cellular kinship. Those properties can be tested statistically using a permutation-based framework.

Simultaneous Assessment of Kinship, Division Number, and Phenotype via Flow Cytometry for Hematopoietic Stem and Progenitor Cells

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

This article outlines the protocol for quantifying the percentage of Tissue Factor (TF)-positive platelets using whole blood flow cytometry, assessing the protein: (1) intracellularly in resting conditions, and (2) on the cell surface, in both resting and activated conditions. Guidance is also provided for evaluating TF-positive platelets in platelet-rich plasma.

Flow Cytometry Analysis of Tissue Factor Expression in Human Platelets

The present protocol provides a comprehensive set of procedures necessary to analyze procoagulant platelets, which exhibit overlapping features of necrosis, apoptosis, and platelet activation.

Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Learn multi-color flow cytometry to identify platelet subpopulations and analyze activation patterns with various agonists. This method reveals distinct clusters for studying thrombosis and inflammation.

Analyzing Platelet Subpopulations by Multi-color Flow Cytometry

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