The authors wish to thank Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert for technical assistance. This work has been supported by ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 and ANR-18-CE14-0037.

All animal experiments were performed in accordance with European standards 2010/63/EU and the CREMEAS Committee on the Ethics of Animal Experiments of the University of Strasbourg (Comit&#233; R&#233;gional d&#39;Ethique en Mati&#232;re d&#39;Exp&#233;rimentation Animale Strasbourg).
1. Preparation of reagents
<ol>
	<li>Prepare reagents as described in Table 1.
	<ol>
		<li>For the Stock I, dissolve each powder separately. Ensure that the osmolarity of the preparation is higher than 295 mOsm/L. This solution can be stored at 4 &#176;C for one year.</li>
		<li>For the Tyrode&#39;s buffer preparation, make the solution as described in Table 1, adjust the volume to 100 mL with distilled water and add 0.1 g anhydrous D (+) sucrose. Adjust to pH 7.3 using 1 N HCl as needed and the osmolarity to 295 mOsm/L. To prevent bacterial growth, add penicillin G at 10 U/mL final concentration and streptomycin sulfate at 0.29 mg/mL final concentration. Filter the final solution through 0.22 &#181;m pores.</li>
	</ol>
	</li>
</ol>
2. Preparation of the experimental set up
<ol>
	<li>On the day of the experiment, warm the Tyrode&#39;s buffer at 37 &#176;C, and turn on the heating chamber of the microscope to bring the temperature to 37 &#176;C.</li>
	<li>Prepare all the necessary tools, such as a timer, incubation chambers, 5 mL syringes, 21 G, forceps, razor blade, Pasteur pipettes, glass slides, and 15 mL centrifuge tube (Figure 1A).</li>
</ol>
3. Isolation of the mouse bone marrow
<ol>
	<li>Euthanize C57BL/6 mice aged 8-12 weeks by CO2 asphyxiation and cervical dislocation. This should be done quickly by a competent and qualified person.</li>
	<li>To avoid microbial contamination, soak the mouse body in 70% (v/v) ethanol before removing the femurs. Use instruments disinfected in 70% ethanol for the dissection. Collect the two femurs and clean them by removing any adherent tissue. After rapid immersion in ethanol, place them in 15 mL centrifuge tube containing 2 mL Tyrode&#39;s buffer.</li>
	<li>Cut away the epiphyses using a sharp razor blade and flush the bone marrow using a 5 mL syringe filled with 2 mL Tyrdode&#39;s buffer. To achieve this, introduce a 21 G needle into the opening of the femur (on the knee side) and slowly press the plunger to retrieve an intact marrow cylinder (Figure 1B,C). Keep the marrow collection session as brief as possible (under 10 min).</li>
</ol>
4. Bone marrow sectioning and placement into the incubation chamber
<ol>
	<li>Use a 3 mL plastic pipette to carefully and gently transfer the intact bone marrow onto a glass slide. It is important that the samples are covered with buffer to prevent drying out (Figure 1D). 
	NOTE: Avoid flow-reflow to minimize shears that could dissociate the tissue.</li>
	<li>Under a stereomicroscope (10x), cut off the ends of the marrow that may have been compressed at the time of the flush. Then cut transversal sections with a sharp razor blade. Sections should be thin enough to allow a detailed observation but ensure that megakaryocytes are not damaged by compression (usually thickness around 0.5 mm) (Figure 1E,F). 
	NOTE: The sections are made with a sharp razor blade and under a magnifier to adjust their thickness to about 0.5 mm. Only the sections of uniform thickness are selected. This procedure is not complicated but its standardization requires some experience.</li>
	<li>Using a plastic pipette, collect 10 sections into 1 mL tube containing Tyrode&#39;s buffer (Figure 1G).</li>
	<li>Carefully transfer the sections to an incubation chamber with a diameter of 13 mm (Figure 1H).</li>
	<li>Aspirate the buffer and adjust the volume to 30 &#181;L of Tyrode&#39;s buffer supplemented with 5 % mouse serum. 
	NOTE: It is at this stage that pharmacological agents can be added to evaluate their impact on proplatelet formation.</li>
	<li>Position the sections at distance. Seal the self-adhesive chamber with a coverslip of the dimension 22 x 55 mm. Incline the coverslip while sticking to avoid the formation of air bubbles (Figure 1I).</li>
	<li>Place the chamber in the heating chamber at 37 &#176;C. From this moment, the chronometer is started (T= 0 h). The experiment runs for 6 h at 37 &#176;C (T= 6 h).</li>
</ol>
5. Real-time observation of marrow explants
<ol>
	<li>Use an inverted phase contrast microscope (40x lens for magnification) coupled to a video camera to observe the bone marrow explants. Let the cells incubate for 30 min before starting the observation. While observing, it is necessary to adjust the focus because megakaryocytes are moving. 
	NOTE: Other observation modes are possible (e.g., DIC, fluorescence using mice whose megakaryocytes express an endogenous fluorescent marker), but phase contrast microscopy is optimal to clearly visualize the long and thin megakaryocyte extensions, allowing precise quantification. After 30 min, the marrow cells gradually migrate to the periphery of the explant, forming a monolayer. After 1 h of incubation, megakaryocytes can be identified by their large size and polylobulated nuclei (Figure 1J,K). After 3 h of incubation, the number of megakaryocytes increases and some have long extensions.</li>
	<li>Make videos to record the transformation of the megakaryocytes.</li>
</ol>
6. Quantification of the proplatelet-extending megakaryocytes
<ol>
	<li>Draw a map to localize each section in the incubation chamber (Figure 1K).</li>
	<li>After 1 h, identify the visible megakaryocytes (i.e., giant polylobulated cells) on each section&#39;s periphery and plot their positions on the drawing. Repeat this procedure after 3 and 6 h. 
	NOTE: On the basis of the drawing, each megakaryocyte can be easily located over time and the evolution of their morphology analyzed (e.g., size, deformation, proplatelet extension, etc.). Another possibility is the use of a specific navigation software.</li>
</ol>

<ol>
	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li><li>Becker, R. P., De Bruyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation:+A+scanning+electron+microscopic+investigation.">The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: A scanning electron microscopic investigation.</a> American Journal of Anatomy. 145 (2), 183-205 (1976).</li><li>Muto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells.">A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells.</a> Archivum Histologicum Japonicum. 39 (1), 51-66 (1976).</li><li>Cramer, E. M., 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=Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand.">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li><li>Kaushansky, K., 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=Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin.">Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.</a> Nature. 369 (6481), 568-571 (1994).</li><li>Strassel, C., 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=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Aguilar, A., 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=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li><li>Scandola, C., 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=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li><li>Eckly, A., 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+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li><li>Eckly, A., 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=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., 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=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li><li>Ortiz-Rivero, 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=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101 (2018).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li><li>Zhang, Y., 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=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Malara, A., 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=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Balduini, A., 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=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li></ol>

The authors declare no conflicts of interests.

Here we describe a simple and low-cost in vitro method to evaluate the efficiency of megakaryocytes to extend proplatelets which have grown in the bone marrow. The bone marrow explant model for mouse has four main advantages. First, there are no advanced technical skills required. Second, the time needed to obtain megakaryocyte-extending proplatelets is quite short, only 6 hours for the explant method, compared to a minimum of 4 days for a conventional culture method starting from mouse progenitors. Third, given that only a small amount of tissue is needed and that the results obtained are reproducible, it reduces the number of mice needed to a minimum (usually 6 mice per experimental condition), making these experiments economically and ethically efficient. Lastly, but importantly, the strength of this method lies in the use of megakaryocytes that have fully developed in their natural environment, which may prove invaluable in revealing phenotypes that could be masked in vitro by potential artifacts of the culture conditions. This has been previously documented in mice with megakaryocyte-restricted MYH9 inactivation where opposing results have been found on proplatelet formation in in vitro (increased formation)10 and in vivo (decreased formation) differentiated megakaryocytes11. These paradoxical results have been explained by the requirement of myosin IIA for normal megakaryocyte differentiation in a constraint environment, while myosin IIA is dispensable for megakaryocyte differentiation in liquid culture7.
An interesting application of the bone marrow explant model is the possibility to study the impact of genetic mutations or deficiencies in transgenic mice and/or pharmacological agents exclusively on the platelet extension process, without interfering with the differentiation process as in the case of in vitro culture12. The ideal situation is to use the bone marrow of one femur as a treated sample and its counterpart as control. In addition, the use of transgenic mice allowing spontaneous fluorescence in the megakaryocytes facilitates the visualization of the platelet extension process. To visualize fluorescent megakaryocytes, one possibility could be to add fluorescence-labelled antibodies against specific megakaryocyte markers in the culture chamber. Another possibility could be the use of genetically engineered mouse models expressing a fluorescent protein, either specifically in the megakaryocytic lineage such as mice with CD41-labelled YFP already reported in the literature13, or in all cells such as mice where GFP is linked to the N-terminus of non-muscular myosin II-A14 as illustrated in Figure 2D.
This explant method, therefore, provides both qualitative and quantitative information for a better understanding of platelet formation in their natural environment. Noteworthy, although this method is simple and fast it remains complementary to the studies performed using classical liquid culture. Each one brings separate knowledge according to the stages of proliferation, maturation, extension of the platelets and platelet release that one wishes to study. For example, where the explant method gives information on the capacity of extension of proplatelets by megakaryocytes that have grown in a physiological context, in vitro culture provides information on the importance of the bone marrow microenvironment such as the impact of cell ridigity7 or extracellular matrix dependency15. Thus, in vitro megakaryocyte cultures make it possible to modulate the parameters of the microenvironment in terms of stiffness and adhesive proteins7,16. Please refer to the article "Megakaryocyte culture in three-dimensional methylcellulose-based hydrogel to improve cell maturation and study the impact of stiffness and confinement" by J. Boscher et al., presented in this issue for more information.

The bone marrow explant technique was first developed by Thi&#233;ry and Bessis in 1956 to describe the formation of rat megakaryocyte cytoplasmic extensions as an initial event in platelet formation1. Using phase contrast and cinematographic techniques, these authors characterized the transformation of mature round megakaryocytes into &#34;squid-like&#34; thrombocytogenic cells with cytoplasmic extensions showing dynamic movements of elongation and contraction. These arms become progressively thinner until they become filiform with small swellings along the arms and at the tips. These typical megakaryocyte elongations, obtained in vitro and in liquid media, have certain similarities with platelets observed in fixed bone marrow, where megakaryocytes protrude long extensions through the sinusoid walls into the blood circulation2,3. The discovery and cloning of TPO in 1994, allowed to differentiate megakaryocytes in culture able to form proplatelet extensions resembling those described in bone marrow explants4,5,6. However, megakaryocyte maturation is far less efficient in culture conditions, notably the extensive internal membrane network of bone marrow matured megakaryocytes is underdeveloped in cultured megakaryocytes, hampering studies on the mechanisms of platelet biogenesis7,8.
We detail here the bone marrow explant model, based on Thi&#233;ry and Bessis, to follow in real-time proplatelet formation of mouse megakaryocytes, which have fully matured in their native environment, thus circumventing possible in vitro artifacts and misinterpretations. Results obtained in wild-type adult mice are presented to illustrate the ability of megakaryocyte to extend proplatelets, their morphology and the complexity of proplatelets. We also introduce a rapid quantifying strategy for quality validation to ensure data accuracy and robustness during the megakaryocyte recording process. The protocol presented here is the most recent version of the method&#160;published as a book chapter previously9.

<table><tbody><tr><td>5 mL syringes</td><td>Terumo</td><td>SS+05S1</td><td></td></tr><tr><td>21-gauge needles</td><td>BD Microlance</td><td>301155</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>21108</td><td></td></tr><tr><td>Coverwall Incubation Chambers</td><td>Electron Microscopy Sciences</td><td>70324-02</td><td>Depth : 0,2 mm</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H-3375</td><td>pH adjusted to 7.5</td></tr><tr><td>Human serum albumin</td><td>VIALEBEX</td><td>authorized medication : n&amp;deg; 3400956446995</td><td>20% (200mg/mL -100mL)</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>BVBW8448</td><td></td></tr><tr><td>Micro Cover Glass</td><td>Electron Microscopy Sciences</td><td>72200-40</td><td>22 mm x 55 mm</td></tr><tr><td>Microscope</td><td>Leica Microsystems SA, Westlar, Germany</td><td>DMI8 - 514341</td><td>air lens</td></tr><tr><td>microscope camera</td><td>Leica Microsystems SA, Westlar, Germany</td><td>K5 CMS GmbH -14401137</td><td>image resolution : 4.2 megapixel</td></tr><tr><td>Mouse serum</td><td>BioWest</td><td>S2160-010</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>PSG 100x</td><td>Gibco, Life Technologies</td><td>1037-016</td><td>10,000 units/mL penicillin, 10,000 &amp;mu;g/mL streptomycin and 29.2 mg/mL glutamine</td></tr><tr><td>Razor blade</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Sucrose D (+)</td><td>Sigma</td><td>G8270</td><td></td></tr></tbody></table>

proplatelet formation dynamics of mouse fresh bone marrow explants

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thi&#233;ry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 &#176;C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

Qualitative results. At the beginning of the experiment, all cells are compacted in the bone marrow section. It takes 30 min for the cells to become clearly visible at the periphery of the explants. The megakaryocytes are then recognizable by their large size and their evolution can then be studied over time (size, shape, dynamic, proplatelet extension and platelet release) (Figure 2A). Small megakaryocytes have a diameter between 20 and 30 &#181;m and their nuclei are polylobulated while mature round megakaryocytes are larger (&#62; 30 &#181;m in diameter) with an enlarged cytoplasm. A few dark megakaryocytes may be observed (Figure 2C). These represent dead cells whose proportion should not exceed 0.5%. A proportion higher than this value indicates a sample preparation problem. The morphology of the nucleus can be easily visualized by varying the focus.
Quantitative results. Megakaryocytes are counted manually as described in 6.2. and classified according to their morphology at 3 h and 6 h after sealing of the incubation chamber. Figure 2A summarizes the four essential megakaryocytes classes: (1) small MKs, (2) large MKs, (3) MKs with thick extensions, (4) MKs with thin, elongated, and ramified extensions. These later are the typical proplatelet-forming megakaryocytes, with the prominent characteristics of swellings along the proplatelets and the presence of refractive buds at their extremities. With the help of mapping (Figure 1K), their evolution can be followed over time. The results are expressed as a percentage of each class at each observation time. Classically, half of the megakaryocytes visible at the periphery extend proplatelets at 6 hours for wild-type C57BL/6 mouse bone marrow (Figure 2B).
It is possible to follow the fate of round MKs by capturing sequential images over time to image how they form proplatelets (Video 1). Interestingly, when MKs with thick extensions were monitored over a period of 3 h, it was observed that the thick extensions could either detach from the cell body and branch into proplatelets or retract to reform large round MKs.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Reagents</td>
			<td>H2O</td>
		</tr>
		<tr>
			<td rowspan="3">Stock I*</td>
			<td>16 g</td>
			<td>0.4 g</td>
			<td>2 g</td>
			<td>0.116 g</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>KCL</td>
			<td>NaHCO3</td>
			<td>NaH2PO4,</td>
			<td>to 100 mL</td>
		</tr>
		<tr>
			<td>(2.73 M)</td>
			<td>(53.6 mM)</td>
			<td>(238 mM)</td>
			<td>(8.6 mM)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stock II</td>
			<td>2.033 g MgCl2.6H2O (0.1 M)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stock III</td>
			<td>2.19 g&#160; CaCl2.6H2O (0.1 M)</td>
			<td></td>
			<td></td>
			<td></td>
			<td>to 100 mL</td>
		</tr>
		<tr>
			<td>HEPES Stock**</td>
			<td>119 g HEPES* (0.5M)</td>
			<td></td>
			<td></td>
			<td></td>
			<td>to 1 L</td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s Buffer***</td>
			<td>5 mL Stock I</td>
			<td>1 mL Stock II</td>
			<td>2 mL Stock III</td>
			<td>1 mL HEPES Stock</td>
			<td>1.8 mL albumin Stock</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of the Tyrode&#39;s buffer. Each stock solution is indicated in the first column of the table. The composition as well as the amount of reagent (given in grams) required for each stock solution is indicated per row. The catalog number and the company of each reagent are given in the table of essential supplies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62501/62501fig01.jpg" /> 
Figure 1: Photographic representations of the sample preparation method for the bone marrow explant. (A) Experimental setup required for the bone marrow preparation. (B) A 21-gauge syringe-mounted needle is inserted into the bone. (C) The bone marrow is flushed into a tube containing Tyrode&#39;s buffer. (D) The bone marrow is then gently deposited on a glass slide. (E) The extremities of the bone marrow are cut off. (F) The marrow cylinder is cut into ten 0.5 mm thick sections. (G-H) The ten sections are transferred to an incubation chamber and observed at 37 &#176;C using an inverted microscope. (I) Representative photo of an incubation chamber containing the ten sections of bone marrow. (J) Peripheral cells migrate to form a layer in which the megakaryocytes become visible. (K) Example of drawing illustrating the ten explant sections in the incubation chamber as well as the location of the megakaryocytes (X blue mark) that have migrated out of the tissue for each section. The arrow shows a megakaryocyte at the periphery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62501/62501fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62501/62501fig02.jpg" /> 
Figure 2: Morphological classification of megakaryocytes in explants over time. (A) Megakaryocytes are classified as &#34;small&#34;, &#34;large&#34;, with &#34;thick extension&#34; or &#34;proplatelet-extending&#34;. Bars: 50 &#181;m (B) Proportion of megakaryocytes in each class was determined at 1 h, 3 h and 6 h for a total of 1,468 megakaryocytes, showing that the proportion of &#34;small&#34; and &#34;spherical&#34; megakaryocytes decreases with time while, in parallel, the proportion of megakaryocytes extending proplatelets increases (n=6 mice). Typically, in the explants of a WT mouse after 3 h, between 8.3 and 11.5 megakaryocytes are observed per section. The error bar corresponds to the standard error of the mean for each sample. (C) Representative image of a dark megakaryocyte. Bar: 50 &#181;m (D) Representative image of a megakaryocyte with non-muscular myosin II-A labeled with a green fluorescent protein. Bar: 50 &#181;m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62501/62501fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Time-lapse video showing a MK extending proplatelets.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62501/video_WT_201210.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Here, we detail the bone marrow explant method, from sample preparation to microscopic slide analysis, to evaluate the ability of megakaryocytes which have differentiated in their physiological environment to form proplatelets.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Nanyang Technological University

Research

JoVE Journal

Biology

2.5K Views.  Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS. Het algemene doel van deze procedure is om in realtime de vorming van proplatelet uit muis megakaryocyten te volgen, die in hun oorspronkelijke omgeving zijn gerijpt. Deze methode heeft het voordeel dat het eenvoudig, reproduceerbaar en snel is. Men kan veel megakaryocyten analyseren en de vorming van proplatelets in slechts zes uur visualiseren, in plaats van te wachten op de eerste dag van de kweek zoals voor muis megakaryocyten in vitro.Een interessante toepassing van deze methode i...

Video: Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Immunology and Infection

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Bioengineering

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 ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

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.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

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 ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Improved Bone Marrow Injury Model for Intramembranous Bone Repair Studies

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage template, the process of intramembranous ossification involves the direct conversion of skeletal stem and progenitor cells (SSPCs) into bone-forming osteoblasts. There are limited surgical methods to model this process in experimental mice. Here, we have improved upon a bone marrow injury model in mice to facilitate the study of bone repair via intramembranous ossification and to assess postnatal regulators of osteogenesis. This method is highly reproducible and user-friendly, and it allows temporal assessment of new bone formation in a short period (3-7 days post-injury) using micro-computed tomography (&#181;CT) and frozen section histology. Furthermore, the contributions of SSPCs and mature osteoblasts can be readily assessed using a combination of fluorescent reporter mice and this intramembranous bone marrow injury model. In clinical contexts, intramembranous bone formation is relevant for healing critical size defects, stabilized fractures, cortical defects, trauma from tumor resections, and joint replacements.

The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

Long bone injuries heal through either endochondral or intramembranous bone formation pathways. Unlike the endochondral pathway that requires a cartilage ...

Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

Source: Kozicky, L. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56689">Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the activation of bone marrow-derived macrophages in response to bacterial lipopolysaccharide and intravenous immunoglobulin.

Source: Kozicky, L. et al., Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation. J. Vis. Exp. (2017)
In ...

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

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