Name: competitive transplants to evaluate hematopoietic stem cell fitness
Uploaded: 2016-08-31T13:00:00
Description: Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

Vi er takknemlige for Roxann Hetu-Arbour for å få hjelp med figuren design og demonstrasjon av prosedyrene. Forskning i laboratoriet ble støttet av en overgang pris fra Cole Foundation, Discovery gi no. 419226-2012 fra naturvitenskap og Engineering Research Council of Canada (NSERC) og Canada Foundation for innovasjon (CFI Leaders Fund gi no. 31377). KMH er en Chercheur-Boursier Junior for Fonds de recherche du Québec - Santé (FRQS).

Alle prosedyrer beskrevet i denne protokollen er godkjent av den institusjonelle dyreetikk komité og følge den kanadiske Rådet for dyrestell retningslinjer. Merk: For å opprettholde sterile / patogenfrie boforhold, gjennomføre alle prosedyrer som involverer direkte håndtering av levende mus inne i et biologisk sikkerhetskabinett eller en laminær hette. Rengjør eller sterilisere bur, restraining enheter, bolig materialer, chow og vann gitt til dyrene på riktig måte. Bruk kun sterile...

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+determination+of+rare+populations+by+flow+cytometry:+A+hematopoietic+stem+cell+perspective.">Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective.</a> Cytometry Part A. 83A (8), 721-727 (2013).</li><li>Abidin, B. M., Owusu Kwarteng, E., Heinonen, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frizzled-6+Regulates+Hematopoietic+Stem/Progenitor+Cell+Survival+and+Self-Renewal.">Frizzled-6 Regulates Hematopoietic Stem/Progenitor Cell Survival and Self-Renewal.</a> J Immunol. 195 (5), 2168-2176 (2015).</li><li>Heinonen, K. M., Vanegas, J. R., Lew, D., Krosl, J., Perreault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt4+enhances+murine+hematopoietic+progenitor+cell+expansion+through+a+planar+cell+polarity-like+pathway.">Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway.</a> PLoS One. 6 (4), e19279 (2011).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Golde, W. T., Gollobin, P., Rodriguez, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+simple,+and+humane+method+for+submandibular+bleeding+of+mice+using+a+lancet.">A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.</a> Lab Anim (NY). 34 (9), 39-43 (2005).</li><li>Santaguida, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JunB+protects+against+myeloid+malignancies+by+limiting+hematopoietic+stem+cell+proliferation+and+differentiation+without+affecting+self-renewal.">JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal.</a> Cancer Cell. 15 (4), 341-352 (2009).</li><li>Czechowicz, A., Kraft, D., Weissman, I. L., Bhattacharya, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transplantation+via+antibody-based+clearance+of+hematopoietic+stem+cell+niches.">Efficient transplantation via antibody-based clearance of hematopoietic stem cell niches.</a> Science. 318 (5854), 1296-1299 (2007).</li><li>Zhang, C. C., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hematopoietic+stem+cells+change+their+surface+phenotype+during+ex+vivo+expansion.">Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion.</a> Blood. 105 (11), 4314-4320 (2005).</li><li>Benveniste, P., 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=Intermediate-Term+Hematopoietic+Stem+Cells+with+Extended+but+Time-Limited+Reconstitution+Potential.">Intermediate-Term Hematopoietic Stem Cells with Extended but Time-Limited Reconstitution Potential.</a> Cell Stem Cell. 6 (1), 48-58 (2010).</li><li>Fazekasde St Groth, 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+evaluation+of+limiting+dilution+assays.">The evaluation of limiting dilution assays.</a> J Immunol Methods. 49 (2), R11-R23 (1982).</li><li>Louis, I., Heinonen, K. M., Chagraoui, J., Vainio, S., Sauvageau, G., Perreault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signaling+protein+Wnt4+enhances+thymopoiesis+and+expands+multipotent+hematopoietic+progenitors+through+beta-catenin-independent+signaling.">The signaling protein Wnt4 enhances thymopoiesis and expands multipotent hematopoietic progenitors through beta-catenin-independent signaling.</a> Immunity. 29 (1), 57-67 (2008).</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 Transplant. 30 (12), 843-849 (2002).</li><li>Benz, 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=Hematopoietic+Stem+Cell+Subtypes+Expand+Differentially+during+Development+and+Display+Distinct+Lymphopoietic+Programs.">Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs.</a> Cell Stem Cell. 10 (3), 273-283 (2012).</li><li>Eppert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+gene+expression+programs+influence+clinical+outcome+in+human+leukemia.">Stem cell gene expression programs influence clinical outcome in human leukemia.</a> Nat Med. 17 (9), 1086-1093 (2011).</li><li>McIntosh, B. 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Protokollen er beskrevet her er utformet for å evaluere den relative trenings av donor (test) HSCs mot kjente konkurrent HSCs. Situasjonen for konkurranse øker den relative følsomheten til analysen (større sannsynlighet for å detektere moderate reduksjoner i stamcelle kondisjon) og gir en intern kontroll for teknisk effekt av bestråling og injeksjon. Men det bør ikke brukes som et absolutt mål på HSC fitness; en nedgang i konkurranse oppløsning betyr ikke automatisk at HSCs ikke ville gjøre det bra i fravær ...

Hematopoiesis er en regenerativ prosess som sikrer påfyll av blodceller som har gått tapt på grunn av skade, stråling og celledød. Denne prosessen er sikret av blodkreft stamceller (HSC) som i stor grad bor i voksen benmarg. I tillegg kan hematopoetiske stamceller kan brukes for terapeutiske formål i autoimmune forstyrrelser, hematologiske ondartede sykdommer og immunsvikt 1. Det er derfor et behov for å bedre forstå mekanismene som regulerer HSC funksjon, inkludert deres proliferative utvidelse og deres evne til å nå og innpode mottakeren benmarg etter transplantasjon. Selv om nyere studier har rapportert flere celleoverflatemarkører, inkludert SLAM familiemedlemmer CD150 og CD48, til prospektivt berike voksne HSCs og foster HSCs til omtrent 50% renhet 2-4, gull standard mål for funksjonell HSCs forblir en in vivo repopulating analyse for å bestemme deres evne til å reetablere blod cell produksjon i en bestrålt vert fem. In vivo klonal repopulating analysen ble opprinnelig utviklet av Till og McCulloch 6 og har siden blitt videreutviklet og utvidet. Som opprinnelig definert, HSCs sikre livslang blodlegemer gjennom selvfornyelse og differensiering. Overføringen av HSCs inn i et bestrålt mottaker gjør det således mulig for oss å bedømme: deres evne til å differensiere ved analyse av de forskjellige blodcelle linjer (T-lymfocytter, B-lymfocytter, granulocytter, monocytter) og deres evne til selvfornyelse gjennom serietransplantasjon. Analysen vil vanligvis innebære sammenligning av funksjonaliteten og / eller kvantitet av to populasjoner av HSCs, for eksempel celler som kommer fra to mus med forskjellige genotyper eller celler som er blitt behandlet eller ubehandlet med ulike faktorer som kan påvirke den vedlikehold eller utvidelse av HSCs i kultur. Donor kimerisme, eller bidrag overført donor HSCs to blodceller kan deretter bli bestemt ved strømningscytometri-analyse i perifert blod og benmarg ved hjelp av celleoverflatemarkører eller andre metoder som vil skille donorceller fra mottakeren, eller vert. De mest brukte markører er sikkert de to alleler for genet Ptprc eller CD45 leukocytt antigen 7 at vi har valgt for eksemplene nedenfor. Den klonal repopulation Analysen kan være enten konkurrerende eller ikke-konkurrerende. I en ikke-konkurrerende setting, blir kontroll- og test HSCs overført til separate resipientmus og resultatet for hver celletype vil være uavhengig av den andre. I et konkurranseutsatt miljø, er funksjonen av både test og kontroll HSCs målt mot en befolkning på konkurrent HSCs. Protokollen er beskrevet her bruker konkurranse innstillingen, men kan også tilpasses for ikke-konkurransesituasjon. Begge fremgangsmåter har sine fordeler og begrensninger, og vi sammenligner dem i detalj idiskusjon. Vi beskriver også ulike tilnærminger for å sikre egenkapitalen i antall transplanterte HSCs, forklare hvordan å tilpasse analysen for kvantifisering av HSCs ved å begrense fortynning analysen (LDA), og gi eksempler på både vellykkede og mislykkede transplantasjoner for tolkningen av resultatene.

<table border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Microtainer tubes with K2EDTA</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">20G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td>For blood sampling from the mandibular vein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabQuake Shaker rotisserie</td>
			<td style="width:89px;">Thermo&#160; Scientific</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">C415110</td>
			<td>Any other rotating mixer will work as well to prevent coagulation of blood samples</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Purified anti-mouse CD16/CD32 (clone 2.4G2, Fc Block)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">553142</td>
			<td>Alternatively use clone 93 from eBioscience (cat # 14-0161) or Biolegend (cat# 101310)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pe-Cy7-conjugated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">25-0031</td>
			<td>For most flow cytometry antibodies, the clone is important but the colours and companies can vary depending on the available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD19 (clone 1D3)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">12-0193</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">APC-eFluor780 (APC-Cy7 equivalent)-conjugated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">47-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC-conjugate anti-mouse CD45.1 (clone A20)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">2.50</td>
			<td style="width:119px;">11-0453</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">eFluor450-conjugated anti-mouse CD45.2 (clone 104)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.00</td>
			<td style="width:119px;">48-0454</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotinylated anti-human/mouse CD45R (B220) (clone RA3-6B2)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD3e (clone 145-2C11)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse CD11b (clone M1/70)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-0112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse GR1 (clone RB6-8C5)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">1.25</td>
			<td style="width:119px;">13-5931</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated anti-mouse TER119 (clone TER119)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">13-5921</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">V500 streptavidin</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">561419</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-conjugated anti-mouse CD117 (clone 2B8)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">553355</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-Cy7-conjugated anti-mouse Ly6A/E (Sca1) (clone D7)</td>
			<td style="width:89px;">BD Biosciences</td>
			<td style="width:112px;">0.25</td>
			<td style="width:119px;">558162</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PerCP-eFluor710-conjugated anti-mouse CD135 (clone A2F10)</td>
			<td style="width:89px;">eBioscience</td>
			<td style="width:112px;">0.50</td>
			<td style="width:119px;">46-1351</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alexa fluor 647-conjugated anti-mouse CD150 (clone TC15-12F12.2)</td>
			<td style="width:89px;">Biolegend</td>
			<td style="width:112px;">0.63</td>
			<td style="width:119px;">115918</td>
			<td>BD Biosciences and eBioscience do not carry the same clone</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 27G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309623</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1ml tuberculin syringe with 25G needle</td>
			<td style="width:89px;">BD Syringe</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 um cell strainer</td>
			<td style="width:89px;">BD Falcon</td>
			<td style="width:112px;"></td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

competitive transplants to evaluate hematopoietic stem cell fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to reestablish blood cell production for the lifespan of the recipient. This protocol explains how to set up a functional assay to compare the HSC capacities of two different populations of cells, such as bone marrow from mice of two different genotypes, and how to analyze the recipient mice by flow cytometry. The protocol uses HSC equivalents rather than cell sorting for standardization and discusses the advantages and disadvantages of both approaches. We further discuss different variations to the basic protocol, including serial transplants, limiting dilution assays, homing assays and non-competitive transplants, including the advantages and preferred uses of these varied approaches. These assays are central for the study of HSC function and could be used not only for the investigation of fundamental HSC intrinsic aspects of biology but also for the development of preclinical assays for bone marrow transplant and HSC expansion in culture.

En generell beskrivelse av konkurranse transplantasjon innstilling, herunder sekundær transplantasjoner (omtalt nærmere nedenfor) kan finnes i figur 1. En representant analyse for pre-transplantasjon benmarg HSCs kan finnes i figur 2. Mer detaljert informasjon om utelukkelse av dubletter og døde celler kan finnes andre steder ni. Figurene 3 og 4 ...

Watch this Scientific Journal Video about Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness at JoVE.com

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

This protocol provides step-by-step guidelines for setting up competitive mouse bone marrow transplant experiments to study hematopoietic stem/progenitor cell function without prior purification of stem cells by cell sorting.

Konkurransedyktige transplantasjoner skal vurdere stamcelle Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

The overall goal of this experiment is to determine the fitness of bone marrow hematopoietic stem cells in vivo using a competitive transplant assay. So this method can help answer key questions in the field of stem cell biology and hematopoietic cell transplantation about the impact of various manipulations on stem cell self-renewal after transplant. The main advantages of this technique are that it requires a limited number of animals and that it does not involve cell sorting.Though this method can provide insight into stem cell function, it can be also useful in other fields such as immunology, through the use of mixed bone marrow chimeras. The initial development of this technique in the 1960s paved the way for researchers in the field of stem cell biology to explore the identity and function of hematopoietic stem cells in humans and mice. Demonstrating the procedure will be Roxanne Hetu-Arbour, a Master's student in the lab, and Edward Owusu Kwarteng, a PhD student.Begin by placing the mouse inside a biological safety cabinet on a surgical platform and wet the skin and fur with 70%ethanol. Next, take hold of a hind foot and cut the skin up along the leg. Proceed to pull away the skin and cut away the excess muscle.Then, with the scissor blades against the pelvic bones as a counter-force, pull on the leg bone to dislocate the femur. Detach the tibia and the femur from the kneecap, then remove the foot, and remove the remaining bits of muscle and tendons. Then, place the bones in two to three milliliters of sterile PBS in one well of a six-well tissue culture plate.When all of the bones have been harvested, grasp the bones one at a time with a forceps, and insert a 25-gauge needle attached to a one-milliliter syringe filled with sterile PBS into one end of the bone. Depress the plunger, collecting the cells in a 15-milliliter conical tube until the center of each bone is white. Using a 22-gauge needle, dissociate the cells into a single cell suspension by repeatedly passing them through the needle tip.After all of the bone marrow has been collected, filter the cells through a 70-micron nylon strainer and count the resulting cell suspensions. Then, centrifuge the bone marrow cells, remove the supernatant, and dilute the samples to a one times ten to the eighth cells per milliliter concentration in fresh PBS, on ice. To establish donor hematopoietic stem cell equivalents, transfer 30 microliter aliquots of cells into five-milliliter round-bottomed polystyrene tubes and add an equal volume of unlabeled antibody against CD16 and CD32 to the samples for five minutes at room temperature to block any non-specific staining.Next, incubate the cells in 90 microliters of the appropriate fluorochrome conjugated primary antibody master mix on ice, protected from light. After 30 minutes, add two milliliters of staining buffer to the cells and vortex the tubes. Follow this with centrifugation.Decant the supernatants and resuspend the pellets by flicking. Then, label the cells with 10 microliters of fluorochrome conjugated streptavidin on ice for 20 minutes in the dark. At the end of the incubation, wash the cells in two more milliliters of buffer and analyze the samples by flow cytometry.Using one sample as a baseline, establish the estimated hematopoietic stem cell equivalents for each of the samples. After calculating the number of hematopoietic stem cells per sample as outlined in the text, mix the competitor and test hematopoietic stem cells at a one-to-one ratio and adjust the final volume of the cells to 200 microliters per injection in sterile PBS. For adoptive transfer of the hematopoietic stem cells, warm up an irradiated recipient mouse with a heat lamp.Then aspirate 750 microliters of cells into one one-milliliter tuberculin syringe equipped with a 27-gauge needle and remove the bubbles. Next, with the mouse in the appropriate restraining device, wipe the injection site with ethanol. Insert the needle into a lateral tail vein, bevel side up and parallel to the skin, and gently depress the plunger.When all of the cells have been injected, remove the needle and compress the injection site with sterile gauze for a few seconds to stop the bleeding. Then place the mouse in a clean cage. To analyze the peripheral blood, pierce the facial skin near the hairless spot located under the jawbone and collect one to two drops of blood in an EDTA tube.Place the tube in a rotator to prevent clotting while waiting to proceed. Next, mix one milliliter of freshly-prepared, room temperature, red blood cell lysis buffer with the blood and transfer the sample to a five-milliliter, round-bottomed polystyrene tube. After four minutes at room temperature, add four volumes of ice-cold PBS to each tube and cover the opening with a cap.Mix the tube a few times by inversion and immediately place the cells on ice. After centrifugation, decant the clear red supernatants and resuspend the pellets by flicking. Then add two milliliters of staining buffer to the cells and briefly vortex them.Follow this with a a second centrifugation. Resuspend the pellets by flicking and proceed with flow cytometry staining as detailed in the text. The competitor-derived cells should be present in all three of the myeloid, B-lymphoid, and T-lymphoid lineages.The competitor and donor cells are also found within both lineage negative and hematopoietic stem cell bone marrow cell populations. When the donor hematopoietic stem cells are functionally equivalent to the competitor cells, the proportions of the donor and the competitor-derived cells are equal. The residual host cells are typically almost exclusively T lymphocytes, as these cells are more radioresistant.When the donor cells are observed at a much lower proportion of peripheral blood leukocytes than the competitor cells, some aspects of hematopoietic stem cell function are defective, and warrant further investigation. When the donor and competitor cells are both present in low numbers and the host cells represent the majority of peripheral blood cells, the transplant is considered unsuccessful, and no conclusions can be drawn about the relative functionality of the donor versus the competitor cells. Once mastered, the bone marrow preparation and the transplantation steps can be completed in six to seven hours if they are performed properly.While attempting this procedure, it is important to remember to maintain sterile conditions during the cell preparation and the animal manipulation steps. Following this procedure, other methods, like a limiting dilution assay, can be performed to answer additional questions regarding the functional stem cell numbers. After watching this video, you should have a good understanding of how to perform competitive bone marrow transplants.Don't forget that working with irradiation can be extremely hazardous, and that the appropriate precautions should always be taken while performing this procedure.

Research

JoVE Journal

Developmental Biology

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