This work was supported by US National Institutes of Health grants to K. Walsh (HL131006, HL138014, and HL132564), to S. Sano (HL152174), American Heart Association grant to M. A. Evans (20POST35210098), and a Japan Heart Foundation grant to H. Ogawa.

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia.
1. Prior to preconditioning
<ol>
	<li>Place the recipient mice on antibiotic-supplemented water (5 mM sulfamethoxazole, 0.86 mM trimethoprim) ~24 h prior to irradiation. This is necessary to prevent infection, as the immune system will be suppressed following irradiation, and maintained for 2 weeks following irradiation. At this point, supplement mice with a nutritional/hydration gel to encourage feeding and to prevent weight loss and dehydration after irradiation.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61875/61875fig01.jpg" /> 
Figure 1: Images showing various preconditioning setups.&#160;(A) Pie-cage total body irradiation setup using gamma-ray (Cesium-137): The radiation beam comes from the back of the irradiator in the y-axis direction (horizontal radiation). (B) Mouse cage total body irradiation setup using X-ray: The mouse cage is placed in the reflective chamber. The radiation beam comes from the top of the irradiator in the shape of a cone (vertical radiation). The distance from the radiation source to the cage is 530 mm. (C) Adjustable tray in X-ray irradiator: This setup is used for partially shielded irradiation using X-ray. The radiation beam comes from the top of the irradiator in the shape of a cone (vertical radiation). The distance from the radiation source to the tray is 373 mm, and the radius is 250 mm. (D) Thorax-shielding: Anesthetized mice are placed on a tray. The mice are placed inverted to each other in supine positions with arms and legs fully extended. The lower end of the lead-shield is aligned with the xiphisternum bone and the upper end with the thymus. (E) Abdominal-shielding: Anesthetized mice are placed as in the thorax-shielding set-up with the lower end of the lead-shield aligned with the anus and the upper end below the diaphragm. (F) Head-shielding irradiation setup using gamma-ray (Cesium-137): The anesthetized mouse&#8217;s forepaws are taped down and the mouse is placed in a conical restrainer. The black lead-shield (marked) covers the mouse&#8217;s head and ears. The radiation beam comes from the back of the irradiator in the direction of the Y-axis (horizontal radiation). <a href="https://www.jove.com/files/ftp_upload/61875/61875fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preconditioning of recipient mice (optional)
<ol>
	<li>Total body irradiation
	<ol>
		<li>Place recipient mice into a uniformly sliced pie-cage, or a mouse cage in the reflective chamber within the calculated radius to receive same irradiation dose; however, a maximum of 8 mice per pie-cage and 5 mice per mouse cage is recommended to ensure uniform irradiation (Figure 1A,B).</li>
		<li>To achieve complete myeloablation, ensure that recipient mice receive a total radiation dose of 11 Gy in two 5.5 Gy fractions separated by a 4&#8211;24 h interval. 
		NOTE: While optimal engraftment can be obtained by implementing a 4 h interval between fractions, this can be extended to a 24 h interval, which can be helpful when labor and/or the irradiator is unavailable.</li>
	</ol>
	</li>
	<li>Partially shielded irradiation
	<ol>
		<li>Anesthetize the recipient mice by intraperitoneal injection of ketamine (80&#8211;100 mg/kg) and xylazine (5&#8211;10 mg/kg). The restraint of mouse movement is critical to ensure the uniform irradiation and effective protection of the targeting organs during the shielding process.</li>
		<li>For thorax and abdomen shielding, orient the radiation beam of the X-ray irradiator vertically to the mouse (Figure 1C).
		<ol>
			<li>Position the anesthetized mice onto a flat plate, centering the radiation source from above. Place the mice inverted to each other in a supine position with arms and legs fully extended (Figure 1D,E). 
			NOTE: X-ray irradiator facility, for this experiment, allows that two mice at a time can be positioned within the effective radius that allows uniform irradiation. While the effective radius is calculated based on the distance between the radiation source and the tray, the number of animals that can be simultaneously irradiated will depend upon the specific irradiator.</li>
			<li>Fasten the paws of the mice onto the plate using tape to ensure the mice are immobilized during the irradiation procedure. Place lead shielding so that it covers regions that require protection.</li>
			<li>For thorax shielding, prepare the lead shield by measuring the length from the mouse&#8217;s xiphisternum bone to the thymus and calculating the thickness that will provide sufficient protection from the source of irradiation. Place the lead shielding so that the lower end aligns with the xiphisternum bone. The upper end of the lead barrier will fit near the thymus (Figure 1D).</li>
			<li>For abdomen shielding, prepare the lead shield by measuring the length from the mouse&#8217;s anus to the diaphragm and calculating the thickness that will provide sufficient protection from the source of irradiation. Place the lead shielding so that the lower end aligns with the anus. The upper end of the lead shield will fit below the diaphragm (Figure 1E). 
			NOTE: Localizing the lead shield to be consistent among cohorts may reduce some variation with regard to the size of the mice.</li>
		</ol>
		</li>
		<li>For head shielding, orient the radiation beam of the Cesium irradiator horizontally to the mouse.
		<ol>
			<li>Carefully tape the forepaws of an anesthetized mouse to the abdomen. This ensures that the arms get a full dose of irradiation and are not covered by the shield.</li>
			<li>For head shielding, place the mouse in a conical restrainer, which fits inside a lead shield. Once the mouse is inside the conical restrainer, slide the restrainer into the slot within the lead shield (Figure 1F). The lead shield should completely cover the mouse&#8217;s head and ears (~3.2 cm), leaving the rest of the mouse&#8217;s body exposed for irradiation. The position of the restrainer inside the shield can be adjusted to fit different sized animals by sliding it further inside or outside the shield.</li>
			<li>Place mice inside the irradiator, perpendicular to the source for irradiation.</li>
		</ol>
		</li>
		<li>Expose mice to two 5.5 Gy fractions of irradiation (total dose of 11 Gy) separated by a 4&#8211;24 h interval.</li>
		<li>After each irradiation fraction, place the cages with anesthetized mice on heated mats or under red heat lamps to prevent hypothermia and aid in the recovery from anesthesia. 
		NOTE: Caution must be taken to not overheat the anesthetized mouse when using a lamp since they cannot escape the heat. As described above, the positioning of animals and the thickness of lead shield can differ between studies based upon the specific features of the irradiator (radiation type/direction of beam, etc.). Researchers will need to adjust their experiments accordingly.</li>
	</ol>
	</li>
</ol>
3. Bone isolation
NOTE: Ideally, donor mice and recipient mice should be similar in age, and within 8&#8211;12 weeks old. Using at least 3 mice as donors (rather than single donor) is preferred to minimize for heterogeneity (even when using mice with the same genotype). Approximately, 40 million unfractionated bone marrow cells can be obtained from six bones (two femurs, two tibias, and two humeri) of a single mouse. Transplantation of 5 million bone marrow cells to each recipient mouse will typically ensure engraftment.
<ol>
	<li>Euthanize donor mice by cervical dislocation without anesthesia (preferred method to avoid chemical contamination of cells) and place each mouse onto an absorbent pad.</li>
	<li>Disinfect the skin using a 70% ethanol spray.</li>
	<li>Make a small transverse cut in the skin below the rib cage and hold the skin tightly at either side of the incision, tear in opposite directions toward the head and feet. Peel off the skin from all the limbs.</li>
	<li>Cut over the shoulders and the elbow joints, and remove the attached muscles and connective tissues with the aid of a Kimwipe to obtain the humeri.</li>
	<li>Carefully dislocate the hip joints between the femur and hip bones. Use blunt scissors to cut along the femur head and detach the legs. Cut over the knee joint to separate the femur and tibia, and carefully remove the attached muscles and connective tissues with the aid of a Kimwipe to harvest the femur and tibia. 
	NOTE: Pay special attention to keep the bone epiphysis intact during this step. Discard any broken bones due to loss of sterility. Hip bones and spine bones can be collected in addition to the femur, tibia, and humerus. To collect spine bones, a mortar and a pestle can be used to crush the bones into pieces and harvest the bone marrow cells.</li>
	<li>Place the isolated bones from mice of the same genotype into correspondingly 50 mL conical tube containing 20 mL ice-cold sterile PBS, and keep it on ice until further use. Pay special attention to correctly place the bones into tubes with matched genotypes.</li>
	<li>Repeat the above steps for each donor animal changing gloves in between each mouse. Also, clean scissors and other instruments with 70% ethanol between each mouse.</li>
</ol>
4. Bone marrow cell isolation
NOTE: Perform the following steps in a biosafety class II cabinet.
<ol>
	<li>Preparation of tube sets: Make a small hole in the bottom of a sterile 0.5 mL microcentrifuge tube using an 18 G needle and place it into a sterile 1.5 mL microcentrifuge tube, which contains 100 &#956;L of ice-cold sterile PBS at the bottom. 
	NOTE: As only six bones can fit into the 0.5 mL microcentrifuge tube, it is recommended to prepare sufficient tube sets to process all the bones at the same time.</li>
	<li>Aspirate the PBS and transfer the isolated bones onto a sterile 100 mm cell culture dish. Holding each bone using fine forceps, carefully cut the epiphyses off each end using small scissors that were sterilized in an autoclave. Place the cut bones into the prepared tube sets.</li>
	<li>Centrifuge the tubes at 10,000 x g for 35 s at 4 &#176;C.</li>
	<li>After centrifugation, confirm that the bone marrow has been successfully removed from the bones. Bones should appear white and translucent with a relatively large red pellet at the bottom of the 1.5 mL microcentrifuge tube. Discard the 0.5 mL microcentrifuge tube. 
	NOTE: If the visual inspection fails to detect bone marrow at the bottom of the 1.5 mL tube, cut the bone again and repeat step 4.3.</li>
	<li>Resuspend the bone marrow in 1 mL of ice-cold PBS, then transfer the cell suspension from the same genotype to a matched 50 mL conical tube.</li>
	<li>Dissociate the cells by passing them through an 18 G needle with a 10 mL syringe 10 times.</li>
	<li>Filter single cell suspensions through a 70 &#956;m cell strainer. Add additional ice-cold PBS to a final volume of 10 mL, and resuspend the cells through the gentle use of pipette-aid.</li>
	<li>Centrifuge at 310 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet with 10 mL of serum-free RPMI media. Spare 30 &#956;L of this material for cell counting.</li>
	<li>Determine cell concentration with a cell counter, and calculate the volume of cell suspension required for the transplantation. For the example of a 100% BMT, 5 x 106 bone marrow cells are required for each recipient mouse. 
	NOTE: For a competitive BMT, prepare a total of 5 x 106 bone marrow cells comprising a mixture of donor cells (e.g., CD45.2+) and competitor cells (e.g., CD45.1+). Preparing extra bone marrow cells is highly recommended. For example, if there are 10 recipient mice per experimental group, we typically prepare enough cells for 12 recipient mice.</li>
	<li>Transfer the calculated volume of cell suspension into a new 50 mL conical tube. Centrifuge at 310 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the cells using the calculated amount of serum-free RPMI medium to achieve the appropriate cell density and volume. Typically, 200 &#956;L is the optimal volume for a retro-orbital injection.</li>
</ol>
5. Transplantation of bone marrow cells to irradiated mice
<ol>
	<li>Anesthetize the recipient mice with 5% isoflurane.</li>
	<li>While mice are anesthetized, slowly inject 200 &#956;L of bone marrow cells into the retro-orbital vein using a 28&#8211;30 G needle with an insulin syringe.
	<ol>
		<li>Alternatively, perform the delivery of the donor cells by tail vein intravenous injection and femoral intramedullary injection, with a maximum volume of 0.2 mL and 25 &#956;L, respectively.</li>
	</ol>
	</li>
	<li>Once the cells are injected, place a drop of proparacaine-containing eye-drops onto the surface of the eye for pain relief. The animal can then be allowed to regain consciousness.</li>
</ol>
6. Transplantation of bone marrow cells to non-conditioned mice
<ol>
	<li>Anesthetize the recipient mice by inhalation of 5% isoflurane.</li>
	<li>Inject 5 x 106 unfractionated bone marrow cells from either genotype retro-orbitally into non-irradiated recipient mice with 28&#8211;30 G insulin syringe.</li>
	<li>Repeat steps 6.1 and 6.2 over 3 consecutive days, such that the recipient mice will be transplanted with a total of 1.5 x 107 bone marrow cells. 
	NOTE: Because the adoptive BMT without pre-conditioning procedure requires bone marrow transplantation for 3 consecutive days, one should attempt to alternate eyes for each injection.</li>
	<li>Post-injection, administer a drop of proparacaine-containing eye drops to the affected eye.</li>
</ol>

<ol>
	<li>Evans, M. A., Sano, S., Walsh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease,+aging,+and+clonal+hematopoiesis.">Cardiovascular disease, aging, and clonal hematopoiesis.</a> Annual Review of Pathology: Mechanisms of Disease. 15 (1), 419-438 (2020).</li><li>Welch, J. 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=The+origin+and+evolution+of+mutations+in+acute+myeloid+leukemia.">The origin and evolution of mutations in acute myeloid leukemia.</a> Cell. 150 (2), 264-278 (2012).</li><li>Jaiswal, 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=Age-related+clonal+hematopoiesis+associated+with+adverse+outcomes.">Age-related clonal hematopoiesis associated with adverse outcomes.</a> New England Journal of Medicine. 371 (26), 2488-2498 (2014).</li><li>Dorsheimer, 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=Association+of+mutations+contributing+to+conal+hematopoiesis+with+prognosis+in+chronic+ischemic+heart+failure.">Association of mutations contributing to conal hematopoiesis with prognosis in chronic ischemic heart failure.</a> JAMA Cardiology. 4 (1), 25 (2019).</li><li>Genovese, 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=Clonal+hematopoiesis+and+blood-cancer+risk+inferred+from+blood+DNA+sequence.">Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.</a> New England Journal of Medicine. 371 (26), 2477-2487 (2014).</li><li>Jaiswal, 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=Clonal+hematopoiesis+and+risk+of+atherosclerotic+cardiovascular+disease.">Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.</a> New England Journal of Medicine. 377 (2), 111-121 (2017).</li><li>Bick, A. 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=Genetic+interleukin+6+signaling+deficiency+attenuates+cardiovascular+risk+in+clonal+hematopoiesis.">Genetic interleukin 6 signaling deficiency attenuates cardiovascular risk in clonal hematopoiesis.</a> Circulation. 141 (2), 124-131 (2020).</li><li>Fuster, J. J., 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=Clonal+hematopoiesis+associated+with+TET2+deficiency+accelerates+atherosclerosis+development+in+mice.">Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice.</a> Science. 355 (6327), 842-847 (2017).</li><li>Wang, 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=Tet2-mediated+clonal+hematopoiesis+in+nonconditioned+mice+accelerates+age-associated+cardiac+dysfunction.">Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.</a> JCI Insight. 5 (6), 135204 (2020).</li><li>Sano, 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=Tet2-mediated+clonal+hematopoiesis+accelerates+heart+failure+through+a+mechanism+involving+the+IL-1&#946;/NLRP3+inflammasome.">Tet2-mediated clonal hematopoiesis accelerates heart failure through a mechanism involving the IL-1&#946;/NLRP3 inflammasome.</a> Journal of the American College of Cardiology. 71 (8), 875-886 (2018).</li><li>Sano, 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=JAK2-mediated+clonal+hematopoiesis+accelerates+pathological+remodeling+in+murine+heart+failure.">JAK2-mediated clonal hematopoiesis accelerates pathological remodeling in murine heart failure.</a> JACC: Basic to Translational Science. 4 (6), 684-697 (2019).</li><li>Yu, K. R., 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=The+impact+of+aging+on+primate+hematopoiesis+as+interrogated+by+clonal+tracking.">The impact of aging on primate hematopoiesis as interrogated by clonal tracking.</a> Blood. 131 (11), 1195-1205 (2018).</li><li>Sano, 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=CRISPR-mediated+gene+editing+to+assess+the+roles+of+Tet2+and+Dnmt3a+in+clonal+hematopoiesis+and+cardiovascular+disease.">CRISPR-mediated gene editing to assess the roles of Tet2 and Dnmt3a in clonal hematopoiesis and cardiovascular disease.</a> Circulation Research. 123 (3), 335-341 (2018).</li><li>Stachura, D. 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=Clonal+analysis+of+hematopoietic+progenitor+cells+in+the+zebrafish.">Clonal analysis of hematopoietic progenitor cells in the zebrafish.</a> Blood. 118 (5), 1274-1282 (2011).</li><li>Gyurkocza, B., Sandmaier, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+regimens+for+hematopoietic+cell+transplantation:+one+size+does+not+fit+all.">Conditioning regimens for hematopoietic cell transplantation: one size does not fit all.</a> Blood. 124 (3), 344-353 (2014).</li><li>Bacigalupo, 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=Defining+the+intensity+of+conditioning+regimens:+working+definitions.">Defining the intensity of conditioning regimens: working definitions.</a> Biology of Blood and Marrow Transplantation. 15 (12), 1628-1633 (2009).</li><li>Abbuehl, J. P., Tatarova, Z., Held, W., Huelsken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+engraftment+of+primary+bone+marrow+stromal+cells+repairs+niche+damage+and+improves+hematopoietic+stem+cell+transplantation.">Long-term engraftment of primary bone marrow stromal cells repairs niche damage and improves hematopoietic stem cell transplantation.</a> Cell Stem Cell. 21 (2), 241-255 (2017).</li><li>Shao, 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=Total+body+irradiation+causes+long-term+mouse+BM+injury+via+induction+of+HSC+premature+senescence+in+an+Ink4a-+and+Arf-independent+manner.">Total body irradiation causes long-term mouse BM injury via induction of HSC premature senescence in an Ink4a- and Arf-independent manner.</a> Blood. 123 (20), 3105-3115 (2014).</li><li>Cui, Y. Z., 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=Optimal+protocol+for+total+body+irradiation+for+allogeneic+bone+marrow+transplantation+in+mice.">Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.</a> Bone Marrow Transplantation. 30 (12), 843-849 (2002).</li><li>Koch, 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=Establishment+of+early+endpoints+in+mouse+total-body+irradiation+model.">Establishment of early endpoints in mouse total-body irradiation model.</a> PLOS One. 11 (8), 0161079 (2016).</li><li>Ismaiel, A., Dumitra&#351;cu, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+risk+in+fatty+liver+disease:+the+liver-heart+axis-literature+review.">Cardiovascular risk in fatty liver disease: the liver-heart axis-literature review.</a> Frontiers in Medicine. 6, 202 (2019).</li><li>Amann, K., Wanner, C., Ritz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk+between+the+kidney+and+the+cardiovascular+system.">Cross-talk between the kidney and the cardiovascular system.</a> Journal of the American Society of Nephrology. 17 (8), 2112-2119 (2006).</li><li>Liao, X., 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=Distinct+roles+of+resident+and+nonresident+macrophages+in+nonischemic+cardiomyopathy.">Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy.</a> Proceedings of the National Academy of Sciences. 115 (20), 4661-4669 (2018).</li><li>Honold, L., Nahrendorf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+and+monocyte-derived+macrophages+in+cardiovascular+disease.">Resident and monocyte-derived macrophages in cardiovascular disease.</a> Circulation Research. 122 (1), 113-127 (2018).</li><li>Lavine, K. J., 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=The+macrophage+in+cardiac+homeostasis+and+disease.">The macrophage in cardiac homeostasis and disease.</a> Journal of the American College of Cardiology. 72 (18), 2213-2230 (2018).</li><li>Ginhoux, F., Guilliams, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophage+ontogeny+and+homeostasis.">Tissue-resident macrophage ontogeny and homeostasis.</a> Immunity. 44 (3), 439-449 (2016).</li><li>Mildner, 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=Microglia+in+the+adult+brain+arise+from+Ly-6C+hi+CCR2++monocytes+only+under+defined+host+conditions.">Microglia in the adult brain arise from Ly-6C hi CCR2+ monocytes only under defined host conditions.</a> Nature Neuroscience. 10 (12), 1544-1553 (2007).</li><li>Cronk, J. 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=Peripherally+derived+macrophages+can+engraft+the+brain+independent+of+irradiation+and+maintain+an+identity+distinct+from+microglia.">Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.</a> Journal of Experimental Medicine. 215 (6), 1627-1647 (2018).</li><li>Lu, R., Czechowicz, A., Seita, J., Jiang, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal-level+lineage+commitment+pathways+of+hematopoietic+stem+cells+in+vivo.">Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo.</a> Proceedings of the National Academy of Sciences. 116 (4), 1447-1456 (2019).</li><li>Gibson, B. W., 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=Comparison+of+cesium-137+and+X-ray+irradiators+by+using+bone+marrow+transplant+reconstitution+in+C57BL/6J+mice.">Comparison of cesium-137 and X-ray irradiators by using bone marrow transplant reconstitution in C57BL/6J mice.</a> Comparative Medicine. 65 (3), 165-172 (2015).</li><li>Cui, Y. Z., 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=Optimal+protocol+for+total+body+irradiation+for+allogeneic+bone+marrow+transplantation+in+mice.">Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice.</a> Bone Marrow Transplantation. 30 (12), 843-849 (2002).</li><li>Kim, C. K., Yang, V. W., Bialkowska, A. B. <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+intestinal+stem+cells+in+epithelial+regeneration+following+radiation-induced+gut+injury.">The role of intestinal stem cells in epithelial regeneration following radiation-induced gut injury.</a> Current Stem Cell Reports. 3 (4), 320-332 (2017).</li></ol>

The authors have nothing to disclose.

For studies of clonal hematopoiesis, we described three methods of BMT: BMT with total-body irradiation, BMT with irradiation with partial shielding, and a less commonly used BMT method that involves no pre-conditioning (adoptive BMT). These methods have been used to assess the impact of clonal hematopoiesis on cardiovascular disease. Researchers can modify these methods accordingly to suit the specific purpose of their study.
Clonal hematopoiesis models 
Clonal hematopoi...

Clonal hematopoiesis (CH) is a condition which is frequently observed in elderly individuals and occurs as a result of an expanded hematopoietic stem and progenitor cell (HSPC) clone carrying a genetic mutation1. It has been suggested that by the age of 50, most individuals will have acquired an average of five exonic mutations in each HSPC2, but most of these mutations will result in little or no phenotypic consequences to the individual. However, if by chance one of these mutations confers a competitive advantage to the HSPC&#8212;such as by promoting it&#8217;s proliferation, self-renewal, survival, or some combination of these&#8212;this may lead to the preferential expansion of the mutant clone relative to the other HSPCs. As a result, the mutation will increasingly spread through the hematopoietic system as the mutated HSPC gives rise to mature blood cells, leading to a distinct population of mutated cells within the peripheral blood. While mutations in dozens of different candidate driver genes have been associated with clonal events within the hematopoietic system, among these, mutations in DNA methyltransferase 3 alpha (DNMT3A) and ten eleven translocation 2 (TET2) are the most prevalent3. Several epidemiological studies have found that individuals who carry these genetic mutations have a significantly higher risk of cardiovascular disease (CVD), stroke, and all-causal mortality3,4,5,6,7. While these studies have identified that an association exists between CH and increased incidence of CVD and stroke, we do not know whether this relationship is causal or a shared epiphenomenon with the aging process. To gain a better understanding of this association, proper animal models that correctly recapitulate the human condition of CH are required.
Several CH animal models have been established by our group and others using zebrafish, mice, and non-human primates8,9,10,11,12,13,14. These models often use hematopoietic reconstitution methods by transplantation of genetically modified cells, sometimes using Cre-lox recombination or the CRISPR system. This approach allows for the analysis of a specific gene mutation in hematopoietic cells to assess how it contributes to disease development. In addition, these models often employ congenic or reporter cells to distinguish the effects of mutant cells from normal or wild-type cells. In many cases, a pre-conditioning regimen is required to successfully engraft donor hematopoietic stem cells.
Currently, the transplantation of bone marrow to recipient mice can be divided into two main categories: 1) myeloablative conditioning and 2) non-conditioned transplantation. Myeloablative conditioning can be achieved by one of two methods, namely, total body irradiation (TBI) or chemotherapy15. TBI is carried out by subjecting the recipient to a lethal dose of gamma or X-ray irradiation, generating DNA breaks or cross-links within rapidly dividing cells, rendering them irreparable16. Busulfan and cyclophosphamide are two commonly used chemotherapy drugs that disrupt the hematopoietic niche and similarly cause DNA damage to rapidly dividing cells. The net result of myeloablative preconditioning is apoptosis of hematopoietic cells, which destroys the recipient&#8217;s hematopoietic system. This strategy not only allows for the successful engraftment of the donor HSPCs, but can also prevent graft rejection by suppressing the recipient&#8217;s immune system. However, myeloablative preconditioning has severe side effects such as damage to tissues and organs and their resident immune cells as well as destruction of the native bone marrow niche17. Therefore, alternative methods have been proposed to overcome these undesirable side effects, particularly in regard to damage to the organs of interest. These methods include shielded irradiation of recipient mice and the adoptive BMT to non-conditioned mice9,17. Shielding the thorax, abdominal cavity, head or other regions from irradiation by the placement of a lead barriers keeps tissues of interest protected from the damaging effects of irradiation and maintains their resident immune cell population. On the other hand, the adoptive BMT of HSPCs to non-conditioned mice has an additional advantage because it preserves the native hematopoietic niche. In this manuscript, we describe the protocols and results of HSPC engraftment after several transplantation regimens in mice, specifically the delivery of HSPC to TBI mice, to mice partially shielded from irradiation, and to non-conditioned mice. The overall goal is to help researchers understand the different physiological effects of each method as well as how they affect experimental outcomes in the setting of CH and cardiovascular disease.

<table>
	<tbody>
		<tr>
			<td>0.5ml microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-408-121</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>1/2 cc LO-DOSE INSULIN SYRINGE</td>
			<td>EXELINT</td>
			<td>26028</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 prfof)</td>
			<td>Fisher chemical</td>
			<td>200559</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>BD 1mL Tuberculin Syringes 25G 5/8 Inch Needle</td>
			<td>Becton Dickinson</td>
			<td>309626</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 18G (1.22mm X 25mm)</td>
			<td>Becton Dickinson</td>
			<td>395195</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Cesium-137 Irradiator</td>
			<td>J. L. Shepherd&#160;</td>
			<td>Mark IV</td>
			<td>equipment</td>
		</tr>
		<tr>
			<td>DietGel 76A</td>
			<td>Clear H2O</td>
			<td>70-01-5022</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Falcon 100 mm TC-Treated Cell Culture Dish</td>
			<td>Life Sciences</td>
			<td>353003</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Fisherbrand sterile cell strainers, 70 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>Isothesia (Isoflurane) solution</td>
			<td>Henry Schein</td>
			<td>29404</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Zoetis</td>
			<td>043-304</td>
			<td>injection</td>
		</tr>
		<tr>
			<td>Kimwipes Delicate Task Wipers</td>
			<td>Kimtech Science</td>
			<td>KCC34155</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>PBS pH7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>RadDisk &#8211; Rodent Irradiator Disk</td>
			<td>Braintree Scientific</td>
			<td>IRD-P M</td>
			<td>general supply</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 (1X)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Sulfamethoxazole and Trimethoprim</td>
			<td>TEVA</td>
			<td>0703-9526-01</td>
			<td>injection</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>139-236</td>
			<td>injection</td>
		</tr>
		<tr>
			<td>X-ray irradiator</td>
			<td>Rad source</td>
			<td>RS-2000</td>
			<td>equipment</td>
		</tr>
	</tbody>
</table>

bone marrow transplantation procedures in mice to study clonal hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

To compare the effect of three BMT/pre-conditioning methods on donor cell engraftment, the fractions of donor cells in peripheral blood and heart tissue were analyzed by flow cytometry at 1-month post-BMT. Isolated cells were stained for specific leukocyte markers to identify the different subsets of leukocytes. In these experiments, wild-type (WT) C57BL/6 (CD45.2) donor bone marrow cells were delivered to WT B6.SJL-PtprcaPrpcb/BoyJ (CD45.1) reci...

Watch this Scientific Journal Video about Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis at JoVE.com

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

bone-marrow-transplantation-procedures-mice-to-study-clonal

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Immunology and Infection

Unit 1

Blood

SCIENTISTS IN ACTION

11.9K Views.  University of Virginia School of Medicine. We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Video: Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...