Name: purification of platelets from mouse blood
Uploaded: 2019-05-07T15:00:00
Description: Watch this Scientific Journal Video about Purification of Platelets from Mouse Blood at JoVE.com

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 bloo...

<ol>
	<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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+blood+platelets+in+tumor+angiogenesis.">The role of blood platelets in tumor angiogenesis.</a> Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 1815 (2), 189-196 (2011).</li><li>Menter, D. 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. M., Ford, T., 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=Isolation+of+the+major+subcellular+organelles+from+mouse+liver+using+Nycodenz+gradients+without+the+use+of+an+ultracentrifuge.">Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge.</a> Analytical Biochemistry. 187 (2), 318-323 (1990).</li><li>Milligan, G. 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=A+lethal+model+of+disseminated+dengue+virus+type+1+infection+in+AG129+mice.">A lethal model of disseminated dengue virus type 1 infection in AG129 mice.</a> Journal of General Virology. 98 (10), 2507-2519 (2017).</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=Lentiviral+gene+rescue+of+a+Bernard-Soulier+mouse+model+to+study+platelet+glycoprotein+Ibbeta+function.">Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibbeta function.</a> Journal of Thrombosis and Haemostasis. 14 (7), 1470-1479 (2016).</li><li>Bergmeier, W., Boulaftali, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+method+to+study+platelet+function+in+mouse+models+of+disease.">Adoptive transfer method to study platelet function in mouse models of disease.</a> Thrombosis Research. 133, S3-S5 (2014).</li><li>Bakchoul, 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=Recommendations+for+the+use+of+the+non-obese+diabetic/severe+combined+immunodeficiency+mouse+model+in+autoimmune+and+drug-induced+thrombocytopenia:+communication+from+the+SSC+of+the+ISTH.">Recommendations for the use of the non-obese diabetic/severe combined immunodeficiency mouse model in autoimmune and drug-induced thrombocytopenia: communication from the SSC of the ISTH.</a> Journal of Thrombosis and Haemostasis. 13 (5), 872-875 (2015).</li><li>Aurbach, K., Spindler, M., Haining, E. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+murine+platelet+structure,+function,+assays,+and+genetic+alterations.">A guide to murine platelet structure, function, assays, and genetic alterations.</a> Journal of Thrombosis and Haemostasis. 5 (4), 661-669 (2007).</li><li>Birschmann, I., 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+functional+highly+purified+human+platelets+for+the+identification+of+new+proteins+of+the+IPP+signaling+pathway.">Use of functional highly purified human platelets for the identification of new proteins of the IPP signaling pathway.</a> Thrombosis Research. 122 (1), 59-68 (2008).</li></ol>

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 &#181;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 &#181;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...

Watch this Scientific Journal Video about Purification of Platelets from Mouse Blood at JoVE.com

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), ...

Platelets should be free from blood cells and proteins when studying their biochemical and physiological properties. Therefore, we have developed a protocol to purify platelets from mouse blood. This technique can purify platelets using iohexol gradient medium and low-speed centrifugation, resulting in a clear separation of platelets from other blood components.To purify the platelets, use a wide-bore pipette tip to layer 200 microliters of freshly harvested whole blood slowly down the side of a 1.5-milliliter tube onto 600 microliters of iohexol gradient medium without mixing. After centrifugation in a swinging-bucket rotor to isolate the platelets, use a new wide-bore pipette tip to collect most of the platelet-rich layer and a small fraction of the platelet-poor layer without aspirating the white blood cell or red blood cell layer. Add the platelet sample to a new tube, and add one milliliter of PBS to the platelets.Mix the platelets by inversion before performing another centrifugation. Then resuspend the pellet in 200 microliters of PBS with mild pipetting. For platelet activation, transfer one to two times 10 to the six platelets to a new tube containing 100 microliters of staining buffer.Add one to two times 10 to the six platelets to a different tube containing 100 microliters of staining buffer supplemented with 0.4-millimolar GPRP peptide. Add thrombin to the tube with the GPRP peptide to activate the platelets, and add the antibody cocktail of interest to both tubes. As a positive control, add one microliter of whole blood in up to 100 microliters of staining buffer in a tube containing 0.4-millimolar GPRP peptide, and add thrombin to activate these platelets.As a negative control, add one microliter of whole blood in up to 100 microliters of staining buffer. Then stain both control tubes with the antibody cocktail. After 30 minutes at room temperature in the dark, wash the samples with one milliliter of fresh staining buffer per tube.Resuspend the pellets in 300 microliters of fresh staining buffer. Then transfer the cell samples into individual flow cytometry analysis tubes protected from light. To analyze the platelets by flow cytometry, create a new experiment, and generate logarithmic forward scatter versus logarithmic side scatter dot plots.After adjusting the voltages as necessary, record the single-color-stained cells for the compensation controls. Now run the positive control sample to acquire enough cells to adjust the compensation and the gates. Then acquire and record 100, 000 events for each whole blood sample, and analyze the flow cytometry data using the appropriate plots and gates.If the blood is added onto the medium slowly, the iohexol gradient medium and the blood sample will form two separate layers in the tube. After centrifugation, the top, straw-colored layer contains the plasma but no intact blood cells. The second, whitish, platelet-rich layer contains the majority of the platelets.The third, transparent layer is the platelet-poor layer and is directly above the red blood cell and white blood cell pellet. Whole blood exhibits distinct populations of red blood cells, white blood cells, and platelets on a logarithmic forward scatter versus side scatter dot plot. Purified platelets, however, demonstrate a distinct population with negligible numbers of red or white blood cells.Whole blood samples contain Ter119-positive red blood cell and CD45-positive white blood cell populations that are nearly absent in purified platelet samples. Untreated purified platelets express almost no activation markers confirming their resting state, whereas purified thrombin-treated platelets typically demonstrate a 71%P-selectin expression. Microscopic evaluation of untreated purified platelet samples reveals no platelet aggregation.After activation with thrombin, however, the platelets demonstrate a robust aggregation, further confirming their viability after purification as demonstrated. Layer the blood sample carefully onto the iohexol medium, and when collecting the platelets, be sure to aspirate carefully as to not collect the red blood cell and white blood cell layers for successful platelet purification. This method can be used for platelet purification from other species as well.The purified platelets can be used for gene expression, activation, aggregation, granule release, and adhesion studies.

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JoVE Journal

Biology

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...