This work was supported by a start-up funding of Cincinnati Children&#8217;s Research Foundation and a University of Cincinnati Pilot Translational grant to M.N.&#160;We would like to thank the Cincinnati Children&#8217;s Hospital Research Flow Cytometry Core for their services.

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
<ol>
	<li>Add 25 &#181;L of 3.2% sodium citrate (pH 7.2) as an anti-coagulant and 0.4 mM of Gly-Pro-Arg-Pro (GPRP) in a polypropylene tube.</li>
	<li>Collect approximately 200 &#181;L of blood from the C57BL/6 mouse using retro-orbital bleeding. 
	NOTE: Blood can be collected from any type of mouse strain regardless of age or gender.
	<ol>
		<li>Use 2 mL of isoflurane in any type of chamber to anesthetize the mouse. 
		NOTE: Depth of anesthesia is checked by signs of increased respiration and decreased movement. The mouse should not respond to movement of the anesthesia chamber, or to being handled. If the mouse is responsive to movement or handling, then it requires more time to keep in the anesthesia chamber.</li>
		<li>1.2.2 Open either right or left eyelid of the mouse and insert a 7.5 cm (0.12 cm diameter) capillary tube into the vascular plexus of the eye.</li>
		<li>Twist the capillary tube one half turn while applying pressure to the capillary tube to break the vessels and initiate blood flow. Hold the animal upside down over the collection vial to be filled by capillary action.</li>
		<li>Once the appropriate volume of blood is collected, remove the capillary tube and close the eye by applying mild pressure for 30 s on the eyelid with gauze. Once bleeding is stopped, return the mouse to its cage and monitor for bleeding.</li>
		<li>Check eyes for trauma 1-2 days after retro-orbital bleeding. Remove any mouse from the study showing signs of significant trauma to the eye as a result of the procedure and euthanize. 
		NOTE: The mouse should not undergo any kind of treatment which can inhibit platelets for at least two weeks before blood collection. The blood sample must be used for platelet purification within one hour of bleeding the mouse.</li>
	</ol>
	</li>
</ol>
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 &#176;C. To prevent platelet degradation, fast pipetting and vigorous shaking should be avoided during the procedure.
<ol>
	<li>Add 600 &#181;L of iohexol gradient medium (12% iohexol powder in 0.85% sodium chloride, 5 mM Tricine, pH 7.2)11 into an 1.5 mL tube.</li>
	<li>Add 200 &#181;L of blood sample slowly down the side of the tube on top of the gradient medium with a wide bore pipette tip so that blood and iohexol medium do not mix (Figure 2A).</li>
	<li>Centrifuge the sample containing tube at 400 x g for 20 min at 20 &#176;C in a swinging bucket rotor with slow acceleration and deceleration.</li>
	<li>Collect most of the platelet-rich layer and a small fraction of platelet-poor layer (total 300 to 400 &#181;L) using a wide bore pipette tip without disturbing the RBC and WBC layers (Figure 2B). Transfer the platelet sample to a new tube. 
	NOTE: If the platelet count is less in the blood or a smaller volume of blood is used, the platelet-rich layer and platelet-poor layer can be seen as a single layer.</li>
	<li>Add 1 mL of PBS to the platelet sample, mix by inverting, and centrifuge at 800 x g for 10 min at 20 &#176;C in a swinging bucket rotor. Discard the solution completely and add 200 &#181;L of PBS to resuspend the platelets with mild pipetting. 
	NOTE: This step is optional, as it removes the gradient medium and plasma proteins and increases the sensitivity of platelets to agonists such as thrombin. Since different centrifuges have different programs, the slow acceleration/deceleration can be determined by reading the user manual of the centrifuge.</li>
	<li>Transfer 30 &#181;L of this sample into a new tube to count platelets. The remaining platelets can be used for biochemical and physiological assays such as platelet activation, aggregation, granule release, etc.</li>
	<li>For RNA or protein extraction, centrifuge the platelet sample at 800 x g for 10 min to pellet the platelets. Discard supernatant completely without disturbing the pellet. Add the desired volume of RNA or protein lysis buffer to lyse the platelets.</li>
</ol>
3. Counting the Platelets
<ol>
	<li>Count the whole blood cells without dilution and purified platelets (diluted 2 to 5-fold with PBS) using an automated cell analyzer. Use 30 to 50 &#181;L samples for counting.</li>
	<li>Calculate the total number of platelets by multiplying by the volume of the sample.</li>
	<li>Calculate the percentage of yield/recovery of purified platelets.</li>
	<li>Count the RBC and WBC in the purified platelet sample (diluted 50 to 100-fold with PBS) with a hemocytometer and microscope.</li>
</ol>
4. Platelet Activation
<ol>
	<li>In a tube, add 1 to 2 million purified platelets in up to 100 &#181;L of staining buffer (PBS with 2% fetal bovine serum, FBS).</li>
	<li>For multiple number of samples, make a master mix of antibodies with 1 &#181;L (0.5 mg/mL) of the following: PE-Cy7 rat anti-mouse TER 119, PE rat anti-mouse CD45, FITC rat anti-mouse CD41, and APC rat anti-mouse/human CD62P (P-selectin) to detect RBC, WBC, platelets, and activated platelets, respectively. Add the master mix to the tubes for staining the cells. Do not add thrombin.</li>
	<li>In another tube, add 1 to 2 million of purified platelets in up to 100 &#181;L of staining buffer, and 0.4 mM GPRP peptide. Add thrombin to activate the platelets and stain the cells following step 4.2. 
	NOTE: GPRP should be added to the sample before adding thrombin to prevent aggregate or clot formation through platelet activation. Thrombin concentration should be optimized to obtain good activation of platelets. After platelet activation, event counts are decreased during acquisition of samples by flow cytometry.</li>
	<li>As a positive control, add 1 &#181;L of whole blood in up to 100 &#181;L staining buffer in a tube and 0.4 mM GPRP peptide. Add thrombin to activate platelets. As a negative control, add 1 &#181;L of whole blood in up to 100 &#181;L of staining buffer. Do not add thrombin in negative control sample. Stain the cells following step 4.2.</li>
	<li>For flow cytometry isotype control, label 5 tubes with unstained, PE-Cy7, PE, FITC, and APC. Add 100 &#181;L of staining buffer, 1 &#181;L of blood, and 1 &#181;L of the respective antibody to the labeled tubes to stain the cells.</li>
	<li>Incubate samples to stain for 30 min at room temperature in the dark.</li>
	<li>Wash the samples by adding 1 mL of staining buffer to each tube. Centrifuge at 800 x g for 5 min at room temperature. Discard the staining buffer carefully without dislodging the pellet.</li>
	<li>Add 300 &#181;L of staining buffer to each tube, mix mildly, and transfer to a FACS tube. Store the stained samples in the dark until analyzed with a flow cytometer.</li>
</ol>
5. Flow Cytometry Analyses of Platelets
<ol>
	<li>Create a new experiment and generate log forward scatter vs log side scatter dot plots and assign gates for Ter119 PE-Cy7 (RBC), CD45 PE (WBC), CD41 FITC (platelets), and CD62P APC (activated platelets) populations according to the gating strategy chosen for the experiment in the FACS DIVA software.
	<ol>
		<li>Open the population hierarchy by choosing Show Population Hierarchy under the Populations tab. Edit the population hierarchy by checking the gating strategy and properly renaming the gates.</li>
		<li>Open statistics view by choosing Create Statistics View. Edit statistics view according to user preferences by right clicking the statistics view and selecting Edit Statistics View.</li>
		<li>Choose a color for each gate that enhances the visual aspect of the layout by double clicking on the box corresponding to the population.</li>
	</ol>
	</li>
	<li>In the Cytometer window, click on the Parameters tab and adjust the voltages for parameters to visualize the population of interest on the plots.</li>
	<li>Adjust voltages for each of the parameters so that the negative population can be distinguished from the positive population. Before recording the samples for the compensation control, check that the single-stained positive controls are on scale (ensure that the positive peaks are visible and not too far to the right). 
	NOTE: If the positive peaks are off scale, the voltages should be adjusted to see the positive populations.</li>
	<li>Record the single-color stained cells for compensation controls (unstained, PE-Cy7, PE, FITC, and APC).</li>
	<li>If the positive control gates are not set up properly, adjust them by changing voltages.</li>
	<li>Go to Experiment &#62; Compensation Setup &#62; Calculate Compensation. Choose Apply Only in the resulting window.</li>
	<li>Switch to the global worksheet by clicking on the toggle button in the top, left of the worksheet window.</li>
	<li>Load the positive control sample and acquire enough cells to adjust the compensation and the gates.</li>
	<li>Load the sample again and check that each plot shows the expected cell population based on the fluorochrome stained antibody. Acquire and record 100,000 events for whole blood samples and 10,000 events for purified platelets. Load the samples one by one until acquisition is completed.</li>
	<li>Analyze the flow cytometry data with the software with appropriate plots and gates (Figure 3)</li>
</ol>

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	<li>de Witt, S. M., Verdoold, R., Cosemans, J. M., Heemskerk, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+platelet-based+control+of+coagulation.">Insights into platelet-based control of coagulation.</a> Thrombosis Research. 133, S139-S148 (2014).</li><li>Machlus, K. R., Thon, J. N., Italiano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+the+developmental+dance+of+the+megakaryocyte:+a+review+of+the+cellular+and+molecular+processes+mediating+platelet+formation.">Interpreting the developmental dance of the megakaryocyte: a review of the cellular and molecular processes mediating platelet formation.</a> British Journal of Haematology. 165 (2), 227-236 (2014).</li><li>Weyrich, A. S., Zimmerman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets:+signaling+cells+in+the+immune+continuum.">Platelets: signaling cells in the immune continuum.</a> Trends in Immunology. 25 (9), 489-495 (2004).</li><li>Nami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+platelets+and+PBMC:+New+evidence+in+wound+healing.">Crosstalk between platelets and PBMC: New evidence in wound healing.</a> Platelets. 27 (2), 143-148 (2016).</li><li>Mancuso, M. E., Santagostino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets:+much+more+than+bricks+in+a+breached+wall.">Platelets: much more than bricks in a breached wall.</a> British Journal of Haematology. 178 (2), 209-219 (2017).</li><li>Hampton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets'+Role+in+Adaptive+Immunity+May+Contribute+to+Sepsis+and+Shock.">Platelets' Role in Adaptive Immunity May Contribute to Sepsis and Shock.</a> Journals of the American Medical Association. 319 (13), 1311-1312 (2018).</li><li>Sabrkhany, S., Griffioen, A. W., Oude Egbrink, M. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+&amp;#34;first+responders&amp;#34;+in+wound+response,+cancer,+and+metastasis.">Platelet &amp;#34;first responders&amp;#34; in wound response, cancer, and metastasis.</a> Cancer and Metastasis Reviews. 36 (2), 199-213 (2017).</li><li>Fink, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+platelet-specific+mRNA+by+real-time+PCR+after+laser-assisted+microdissection.">Characterization of platelet-specific mRNA by real-time PCR after laser-assisted microdissection.</a> Thrombosis and Haemostasis. 90 (4), 749-756 (2003).</li><li>Trichler, S. A., Bulla, S. C., Thomason, J., Lunsford, K. V., Bulla, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-pure+platelet+isolation+from+canine+whole+blood.">Ultra-pure platelet isolation from canine whole blood.</a> BioMed Central Veterinary Research. 9, 144 (2013).</li><li>Ford, T. C., Graham, J., Rickwood, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new,+rapid,+one-step+method+for+the+isolation+of+platelets+from+human+blood.">A new, rapid, one-step method for the isolation of platelets from human blood.</a> Clinica Chimica Acta. 192 (2), 115-119 (1990).</li><li>Noisakran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+bone+marrow+cells+by+dengue+virus+in+vivo.">Infection of bone marrow cells by dengue virus in vivo.</a> Experimental Hematolology. 40 (3), 250-259 (2012).</li><li>Rickwood, D., Ford, T., Graham, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nycodenz:+a+new+nonionic+iodinated+gradient+medium.">Nycodenz: a new nonionic iodinated gradient medium.</a> Analytical Biochemistry. 123 (1), 23-31 (1982).</li><li>Graham, J. 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J., Bender, M., Pleines, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+collection,+platelet+isolation+and+measurement+of+platelet+count+and+size+in+mice-a+practical+guide.">Blood collection, platelet isolation and measurement of platelet count and size in mice-a practical guide.</a> Platelets. , 1-10 (2018).</li><li>Jirouskova, M., Shet, A. S., Johnson, G. 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The authors declare no conflict of interest.

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 gradien...

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&#8217; 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.

<table><tbody><tr><td>APC rat anti-mouse/human CD62P (P-selectin)</td><td>Thermoscientific</td><td>17-0626-82</td><td>Platelets activation marker</td></tr><tr><td>Eppendorf tube</td><td>Fisher Scientific</td><td>14-222-166</td><td>Tube for centifuge</td></tr><tr><td>FACS DIVA software</td><td>BD Biosciences</td><td>Non-catalog item</td><td>Analysis of platelets and whole blood</td></tr><tr><td>FACS tube</td><td>Fisher Scientific</td><td>352008</td><td>Tubes for flow cytomtery</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td>Ingredient for staining buffer</td></tr><tr><td>FITC rat anti-mouse CD41</td><td>BD Biosciences</td><td>553848</td><td>Platelets marker</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>Non-catalog item</td><td>Analysis of platelets and whole blood</td></tr><tr><td>FlowJo software</td><td>FlowJo, Inc.</td><td>Non-catalog item</td><td>Analysis of platelets and whole blood</td></tr><tr><td>Gly-Pro-Arg-Pro (GPRP)</td><td>EMD Millipore</td><td>03-34-0001</td><td>Prevent platelet clot formation</td></tr><tr><td>Hematocrit Capillary tube</td><td>Fisher Scientific</td><td>22-362566</td><td>Blood collection capillary tube</td></tr><tr><td>Hemavet</td><td>Drew Scientific</td><td>Non-catalog item</td><td>Blood cell analyzer</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>3100</td><td>Cell counting chamber</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>1001936040</td><td>Use to Anesthetize mouse</td></tr><tr><td>Microscope (Olympus CKX41)</td><td>Olympus</td><td>Non-catalog item</td><td>Cell monitoring and counting</td></tr><tr><td>Nycodenz (Histodenz)</td><td>Sigma-Aldrich</td><td>D2158</td><td>Gradient medium</td></tr><tr><td>PE rat anti-mouse CD45</td><td>BD Biosciences</td><td>561087</td><td>WBC marker</td></tr><tr><td>PE-Cy7 rat anti-mouse TER 119</td><td>BD Biosciences</td><td>557853</td><td>RBC marker</td></tr><tr><td>Pipet tips 200 &amp;micro;L, wide-bore</td><td>ThermoFisher Scientific</td><td>21-236-1A</td><td>Transferring blood and platelet samples</td></tr><tr><td>Pipet tips 1000 &amp;micro;L, wide-bore</td><td>ThermoFisher Scientific</td><td>21-236-2C</td><td>Transferring blood and platelet samples</td></tr><tr><td>Phosphate buffured saline (PBS)</td><td>ThermoFisher Scientific</td><td>14040-117</td><td>Buffer for washing and dilution</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td>Physiological saline</td></tr><tr><td>Sodium citrate</td><td>Fisher Scientific</td><td>02-688-26</td><td>Anti-coagulant</td></tr><tr><td>Staining buffer</td><td>In-house</td><td>Non-catalog item</td><td>Wash and dilution buffer</td></tr><tr><td>Steile water</td><td>In-house</td><td>Non-catalog item</td><td>Solvent</td></tr><tr><td>Table top centrifuge</td><td>ThermoFisher Scientific</td><td>75253839/433607</td><td>Swinging bucket rotor centrifuge</td></tr><tr><td>Thrombin</td><td>Enzyme Research Laboratoty</td><td>HT 1002a</td><td>Platelet activation agonist</td></tr><tr><td>Tricine</td><td>Sigma-Aldrich</td><td>T0377</td><td>Buffer for Nycodenz medium</td></tr></tbody></table>

purification of platelets from mouse blood

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 &#176;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.

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 &#176;C. The quality of the purified platelets was evaluated with microscopy and flow cytometry after staining with antibody to detect any contaminating cells and acti...

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Purification of Platelets from Mouse Blood

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), ...

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21.9K Views.  Cincinnati Children's Hospital Medical Center. Bloedplaatjes moeten vrij zijn van bloedcellen en eiwitten bij het bestuderen van hun biochemische en fysiologische eigenschappen. Daarom hebben we een protocol ontwikkeld om bloedplaatjes te zuiveren uit muisbloed. Deze techniek kan bloedplaatjes zuiveren met behulp van iohexol gradiënt medium en lage snelheid centrifugatie, wat resulteert in een duidelijke scheiding van bloedplaatjes van andere bloedbestanddelen.Om de bloedplaatjes te zuiveren, gebruik een breedborende pipetpunt om ...

Video: Purification of Platelets from Mouse Blood

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Immunology and Infection

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Bioengineering

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

Medicine

Isolation of Mouse Megakaryocyte Progenitors

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

This method describes the purification by flow cytometry of MEP and MKp from mice femurs, tibias, and pelvic bones.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through ...

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

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

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.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing ...

Analyzing Platelet Phosphatidylserine and Microvesicle Release

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

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

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.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Source: Perdomo, J., et al. <a href="https://app.jove.com/v/56420">Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells &#8212; isolated from human umbilical cord blood &#8212; differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

Source: Perdomo, J., et al. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (2017)
This video ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Source: Sionov, R. V., et al., <a href="https://app.jove.com/v/52933">Isolation and Characterization of Neutrophils with Anti-Tumor Properties.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

Source: Sionov, R. V., et al., Isolation and Characterization of Neutrophils with Anti-Tumor Properties. J. Vis. Exp. (2015)
In this video, we demonstrate ...

Purification of Platelets from Mouse Blood

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