We thank all the members of the Majeti lab for their help, support, encouragement, and inspiration over the years. We acknowledge the Flow Cytometry Core of the Stanford Stem Cell Institute, the Binns Program for Cord Blood Research, and the patients for donating their samples. For human samples, normal donor human bone marrow and peripheral blood cells were obtained fresh from AllCells or the Stanford Blood Center. We thank the Nakauchi lab at Stanford University for donating the pBac-AkaLuc-tdTomato plasmid. Above all, we would like to thank the veterinarians and animal control staff at the Veterinary Service Center at Stanford who take care of our mice. In particular, Mike Alvares, supervisor of the animal center, has been so thorough in his management of the mice that it is no exaggeration to say that without him, our research would not have been possible.
This work was supported by NIH grants 1R01HL142637 and 1R01CA251331, the Stanford Ludwig Center for Cancer Stem Cell Research and Medicine, and the Blood Cancer Discoveries Grant program through The Leukemia &#38; Lymphoma Society, The Mark Foundation for Cancer Research, and The Paul G. Allen Frontiers Group, all to R.M. R.M. is a recipient of a Leukemia and Lymphoma Society Scholar Award. Y.N. was supported by the Nakayama Foundation for Human Science and a Stanford University School of Medicine Dean&#39;s Postdoctoral Fellowship. A.E. was supported by the NCI under award F32CA250304, the Advanced Residency Training Program at Stanford, and the American Society of Hematology Scholar Award.

Six- to ten-week-old male or female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used in this protocol, but it can be applied to all types of mice. The irradiation depends on the experimental content and the type of mice, whether or not to irradiate the mice depends on the research objectives. All animal procedures described here were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by Stanford University&#39;s Administrative Panel on Lab Animal Care (APLAC #22264). All normal blood cell populations were sorted out fresh. Human AML samples were obtained from patients at the Stanford Medical Center with informed consent, according to institutional review board (IRB)-approved protocols (Stanford IRB, 33818).
1. Intrafemoral injection of cell lines, hematopoietic stem and progenitor cells (HSPCs), or leukemic stem cells (LSCs)
NOTE: To easily analyze the injected cells with an imaging device, an Akaluc/tdTomato-positive K562 cell line was generated, and AkaLumine-HCl/Akaluc was used as a substrate10. If imaging equipment is not available, cells can be labeled with a fluorescent dye by lentivirus or PiggyBac and analyzed by flow cytometry. Regardless of the details of cell preparation, having a way to prove that the cells administered into the bone marrow have reliably entered the BM is important for improving this technique (Figure 1).
<ol>
	<li>Prepare cell suspensions (up to 3 &#215; 106 blood cells) with 20 &#181;L of fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS) + 2 mM EDTA) or Thaw medium (IMDM + 20% FBS + Pen/Strep) in PCR or 1.5 mL tubes and keep on ice before injection. 
	NOTE: Avoid making air bubbles while suspending the cells with the needle. Bubbles should be removed as much as possible right before the injection, as injecting air bubbles can cause sudden death of mice.</li>
	<li>Condition 6-10-week-old adult mice with 200 cGy (225 kV, 13.3 mA, total 2 Gy [Dose control mode]) up to 48 h prior to injection of the cells.</li>
	<li>First, anesthetize the mice with isoflurane inhalation in an induction chamber and maintain at 2% isoflurane in 100% oxygen at a flow rate of 1-2 L/min via a nose cone to reach a steady state of anesthesia. Check the depth of anesthesia with a toe pinch reflex and slow, steady breathing, and maintain this state throughout the isoflurane procedure. 
	NOTE: Monitor the depth of the anesthesia every 3-5 min throughout the procedure to ensure there is no change in heart and respiratory rates with surgical manipulation and/or ear, toe, and tail pinch. The color of mucous membranes and skin can be used as an indicator of oxygenation, as they will be pinkish if the mice are in good condition. Exercise caution when opaque drapes are placed over the mouse, as it is not always possible to see what is happening with the mouse.</li>
	<li>Prior to the procedure, administer mice with 10 mg/kg carprofen subcutaneously to minimize pain and warm 0.5-1.0 mL of 0.9% saline for supportive care.</li>
	<li>Apply ophthalmic ointment to the eyes of the mouse to avoid corneal drying.</li>
	<li>Keep the mouse on a heat pad or other thermostatically controlled surface to prevent hypothermia during the procedure. 
	NOTE: After the procedure, a temperature-adjustable heating pad is used. The body temperature of the mouse is typically 37-39 &#176;C, and the pad should ideally be slightly lower than this. In practice, the temperature is often set at 32-36 &#176;C.</li>
	<li>Disinfect the entire leg containing the femur to be injected with three sets of alternating scrubs (alternating with either a povidone-iodine or a chlorhexidine scrub and 70% ethanol-soaked gauze sponges). 
	NOTE: 1) If the operator is left-handed, the left femur of the mouse might be easier to inject than the right femur. 2) In this study, the fur was not removed for injections to avoid unnecessary skin damage, and the needle entry site can be easily located without cutting the fur off. However, in mice with dark fur, it may be challenging to locate the puncture site, in which case, consider cutting off the fur as it is quick and easy to start the injection.</li>
	<li>Pinch the femur gently with the thumb and index fingers to stabilize the leg. Push the tibia with either the ring or the fifth finger to keep the tibia bent from the femur. Positioning is important for successful injection (Supplementary Figure S1-S5). 
	NOTE: Use forceps to pinch the bone to prevent injury to the fingers; however, it might be difficult to feel the shaft of the femur and will add time to the procedure.</li>
	<li>Insert an empty sterile 27 G needle (1/2 inch) with an attached syringe just under the patellar tendon so that the needle is lodged securely between the two condyles of the femur. Use the edge of the needle and peck the patellar tendon on top of the femur lightly a couple of times to find the best place to insert the needle (Supplementary Figure S1-S5). 
	NOTE: A knowledge of the anatomy of the mouse knee area is necessary before performing this procedure. Beginners should take the time to understand the anatomy of the area and practice the procedure on an euthanized mouse before attempting it on a live mouse.</li>
	<li>Swivel the needle outward and upward to ensure it is parallel with the shaft of the femur. 
	NOTE: This maneuver provides a reliable path to the marrow cavity, facilitates retrieval of marrow contents from the femoral shaft, and minimizes discomfort to the animal from the procedure.</li>
	<li>Turn the needle in circles while slowly advancing into the femoral marrow cavity. Insert the needle until there is a noticeable reduction in resistance. Confirm the correct positioning of the needle by gently moving the syringe laterally. If it is possible to touch the edge of the needle with the fingers coming outside the bone, go back to step 1.9 and find another angle and place to drill the needle down.
	<ol>
		<li>Ensure that the needle&#39;s entry angle is perpendicular to the bone end-face. 
		NOTE: Theoretically, the needle must be injected parallel to the femur and must be at a 90&#176; angle to the bone ends. If there is resistance from the interior surface, the needle has been correctly placed in the femoral cavity. To make sure the needle is parallel to the bone, place a light on the side of the bone and observe the shadow of the bone parallel to the shaft of the needle. The decrease in resistance upon entry into the bone marrow cavity depends on the age of the mouse: the younger the mouse, the easier it is to recognize the decrease in resistance.</li>
	</ol>
	</li>
	<li>Create negative pressure by gently pulling the needle plunger back while moving the needle back and forth within the BM cavity. If the needle is in the BM cavity, some blood/marrow will come out in the syringe.
	<ol>
		<li>If the blood/marrow is not seen, change to a new needle and try to find the same route. 
		NOTE: The reason for changing to a new needle is that once the bone clogs the needle, it becomes difficult to check for the backflow of the blood/marrow. Minimizing the number of injection routes into the BM cavity is crucial, as cells injected into the bone marrow have the potential to leak out of the femur through previous entry points. If multiple injection routes are necessary, leaking risk can be reduced by injecting cells gradually rather than a quick bolus.</li>
	</ol>
	</li>
	<li>Remove the needle slowly and insert the cell-containing syringe through the same route. Try to insert the needle until it stops (at the edge of the BM cavity) and pull back a little bit (1-2 mm) from there to inject the cells easily. Prior to depressing the plunger and releasing the cells, aspirate slightly and check for blood/marrow to ensure the correct placement of the needle. Check the backflow of the blood/marrow first and then push the syringe slowly to inject the cells. 
	&#8203;NOTES: It is very important to remember the route and angle of the first injection when switching to a new needle. If the same route cannot be found, try again with the original needle used to make the first route. A new needle may be used at this time, but if the original needle is used, the jammed bone should be pushed out once before use. Do not use the needle with the cells to find a new route to inject; otherwise, pieces of bone stuck in the needle may prevent cell dispersal. Although the BM cavity is very small, the injection volume should be less than 30 &#181;L, considering the dead cavity.
	<ol>
		<li>Ensure that the speed of injection is slow to prevent air in the syringe from entering the bone marrow and to prevent cells from leaking from the puncture site. If resistance is felt while pushing the syringe, move the needle up and down to find a less resistant area in the cavity.</li>
	</ol>
	</li>
	<li>Once the cells are successfully injected into the femur, remove the needle and syringe from the mouse while maintaining pressure on the syringe. 
	NOTE: Try again with the other femur if the injection cannot be completed. The procedure is very stressful for the mouse, even under anesthesia. Always monitor the vital signs (heart and breathing rate) of the mouse and try to finish the procedure as soon as possible.</li>
	<li>Remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding. During the recovery, keep the mouse on a heating pad or other thermostatically controlled surface. Monitor all mice to verify recovery from anesthesia prior to placing them back on the mouse rack. 
	NOTE: There should be no complication or distress post aspiration if done properly. The procedure will be completed when anesthetized mice have recovered and are able to ambulate and reach food and water.</li>
	<li>Observe the mice for signs of distress or infection post-procedure over the next 24 h. Signs of distress or infection include constant bleeding, anemia, and lethargy. If any of these signs are seen post-procedure, euthanize the animal(s) by CO2 inhalation or cervical dislocation according to the animal handling protocol.</li>
</ol>
2. Aspiration of bone marrow cells from the femur for the FACS analysis
NOTE: The procedure for aspirating the BM cells from the mice is very similar to the methods described in section 1 and can be learned from some previous literature9,11,12. The following is an overview of some differences between the BM injection and aspiration protocols.
<ol>
	<li>Prepare 500 &#181;L of PBS + 10 mM EDTA (cell suspension medium: CSM) per sample for suspending the BM aspiration.</li>
	<li>Wet a 0.5 mL 27 G syringe with CSM before aspirating the BM. Fill the syringe with 200-500 &#181;L of CSM and immediately expel it. Repeat this procedure 2-3x.</li>
	<li>Aspirate the BM gently by withdrawing the plunger on the syringe yielding 20-50 &#181;L of mouse BM. Then, fully remove the needle from the femur and suspend the aspirated sample in CSM (500 &#181;L in a 1.5 mL tube) for the following staining step. Remove the mouse from the nose cone and place it on a clean paper towel to prevent aspiration of bedding during recovery. Monitor all mice to verify recovery from anesthesia prior to placing them back on the mouse rack. 
	NOTE: BM aspiration/sampling can be repeated, but the repeat procedure on the same day must be performed on the opposite femur to prevent repeated trauma to the same leg. Although there is little information on the frequency of BM aspiration, we believe that femoral BM aspiration is generally repeated every 4 weeks and can be performed at least 3-4x in total without any issues (e.g., infection) based on our experience. However, it should be kept in mind that the bone marrow on the contralateral side of the transplanted bone marrow generally has lower cellular engraftment.</li>
	<li>Pellet cells from the bone marrow aspirate by a 5 min spin at 300 &#215; g, 4 &#176;C.</li>
	<li>Aspirate the supernatant, add 0.5-1 mL of ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) to each tube, and vortex to resuspend the cells. Place on ice for 5-10 min.</li>
	<li>Filter each tube through nylon mesh into a new tube.</li>
	<li>Rinse the tube with 1 mL of FACS buffer and add it through the nylon mesh into a new tube as well.</li>
	<li>Pellet cells by a 5 min spin at 300 &#215; g, 4 &#176;C.</li>
	<li>Aspirate the supernatant and add the staining solution containing the desired antibodies13. Place on ice for 20 min.</li>
	<li>Wash the cells and analyze them as per the experimental setup.
	<ol>
		<li>Wash cells with FACS buffer (PBS, 2% FBS, 2 mM EDTA) and stain them with antibodies for 30 min on ice in a total volume of 50 &#181;L. Wash and stain the cells with propidium iodide (PI) at a final concentration of 1 &#181;g/mL immediately before the analysis or sorting. Perform post sort analyses to verify the purity of the sorted cell populations. 
		NOTE: All antibodies used for flow cytometry are detailed in the Table of Materials.</li>
		<li>Use the following FACS gating strategies (Figure 2-7) to analyze the engrafted cells from the mouse bone marrow.
		<ol>
			<li>Distinguish populations of cells by their forward (size) and side scatter (granularity) properties.</li>
			<li>Perform doublet discrimination by plotting FSC-H vs FSC-W, following SSC-H vs SSC-W.</li>
			<li>Exclude dead cells.</li>
			<li>Distinguish populations of cells by human CD45 and mouse CD45. These antibody staining combinations enable the detection of human CD3, CD19, and CD33 as well within the human CD45 fraction. 
			NOTE: Before staining the samples, remember to use mouse and human Fc blocks to prevent non-specific antibody binding. We usually stain with the Fc blocks (mouse + human) for 10 min on ice and then add the antibodies without washing the cells. Refer to each company&#39;s instructions on how to use the Fc block antibodies.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Intrafemoral Injection for Bone Marrow Study in Live Mice

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	<li>Majeti, R., Park, C. Y., 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=Identification+of+a+hierarchy+of+multipotent+hematopoietic+progenitors+in+human+cord+blood.">Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood.</a> Cell Stem Cell. 1 (6), 635-645 (2007).</li><li>Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+self-renewal+in+vivo+and+ex+vivo.+Nature+reviews.">Haematopoietic stem cell self-renewal in vivo and ex vivo. Nature reviews.</a> Genetics. 132, 1-14 (2020).</li><li>Pinho, S., Frenette, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+activity+and+interactions+with+the+niche.">Haematopoietic stem cell activity and interactions with the niche.</a> Nature Reviews Molecular Cell Biology. 20 (5), 303-320 (2019).</li><li>M&#233;ndez-Ferrer, 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=Bone+marrow+niches+in+haematological+malignancies.">Bone marrow niches in haematological malignancies.</a> Nature Reviews. Cancer. 20 (5), 285-298 (2020).</li><li>Frassoni, F., 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=Direct+intrabone+transplant+of+unrelated+cord-blood+cells+in+acute+leukaemia:+a+phase+I/II+study.">Direct intrabone transplant of unrelated cord-blood cells in acute leukaemia: a phase I/II study.</a> The Lancet. Oncology. 9 (9), 831-839 (2008).</li><li>Mazurier, F., Doedens, M., Gan, O. I., Dick, 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=Rapid+myeloerythroid+repopulation+after+intrafemoral+transplantation+of+NOD-SCID+mice+reveals+a+new+class+of+human+stem+cells.">Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.</a> Nature Medicine. 9 (7), 959-963 (2003).</li><li>McKenzie, J. L., Gan, O. I., Doedens, M., Dick, 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=Human+short-term+repopulating+stem+cells+are+efficiently+detected+following+intrafemoral+transplantation+into+NOD/SCID+recipients+depleted+of+CD122++cells.">Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells.</a> Blood. 106 (4), 1259-1261 (2005).</li><li>Futrega, K., Lott, W. B., Doran, M. 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R.M. is on the Advisory Boards of Kodikaz Therapeutic Solutions, Orbital Therapeutics, and 858 Therapeutics and is an inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences. R.M. is a co-founder and equity holder of Pheast Therapeutics, MyeloGene, and Orbital Therapeutics.

Murine xenograft models are important for studying both normal and pathological human hematopoiesis. The BM is the source of hematopoiesis3; therefore, studying hematological diseases requires the successful engraftment of rare human stem cells into the murine BM. So far, transplantation methods such as a tail vein, retroorbital vein, and IF injection have been reported, and it is known that any of these methods can engraft transplanted cells. IF injection is a transplantation method that has been...

The blood system is maintained throughout life by hematopoietic stem cells (HSCs), which reside in bone marrow (BM)1,2. To study dynamic changes in the BM environment, it is important to understand the biology of both normal and malignant hematopoiesis3,4. Transplantation of HSCs directly into human BM yields higher engraftment than peripheral blood (PB) infusion, but high procedural complexity and increased risk of infection preclude this method from being part of standard practice5. In mice, procedures that facilitate BM access without sacrificing the animals provide a resource to serially monitor hematopoiesis. The described procedure aims to produce mice with high engraftment of transplanted cells and allow for serial sampling of the BM of live mice. This paper will focus on producing xenograft models using immunodeficient mice engrafted with human cells, which are more challenging to produce than mouse-mouse allotransplantation models. Compared to conventional transplantation of hematopoietic stem and progenitor cells (HSPCs) through the tail or retroorbital vein, the advantages of this procedure are high cellular engraftment with a low amount of starting material.
Although intrafemoral (IF) cellular injection into the BM cavity of mice is commonly used to study human HSPCs in vivo6,7,8, a formal step-by-step procedure illustrating/filming this technique has not been previously published. This protocol enables high engraftment from a low number of transplanted cells and a mechanism to sample the BM serially. Furthermore, it is possible to utilize this method to analyze the effects of injecting drugs directly into the BM cavity on the treatment of blood diseases. The procedure described here helps obtain access to BM where hematopoietic cells reside without sacrificing the mice.
This protocol is similar to the technique used for BM aspirations9. The key difference is that this paper and the accompanying video protocol detail a safe procedure for injecting cells into the marrow, whereas previous papers transplanted cells via the vein and then performed serial BM aspiration. This protocol enables successful engraftment with small numbers of a cell line (Figure 1), normal cord blood (CB)-derived HSPCs (CD34+) (Figure 2) and HSCs (CD34+CD38-CD45RA-CD90+) (Figure 3), normal BM-derived HSPCs (CD34+CD38-) (Figure 4), patient-derived leukemic stem cells (CD34+CD38-) (Figure 5), CRISPR/Cas9 gene-edited normal CB-derived HSPCs (CD34+) (Figure 6), and acute myeloid leukemia (AML)-iPSCs (Figure 7). Especially for the normal cord blood-derived HSCs, we could successfully make engraftment with only 10 cells. This method is especially valuable for experiments performed with rare or difficult-to-generate cell populations such as unmodified or gene-edited primary human acute myeloid leukemia or CB cells. Furthermore, this procedure describes an efficient method for the serial analysis of engrafted cells, which can be immediately used in downstream experiments. To avoid wasting valuable samples and ensure IF injection, we have also briefly described Akaluc-based procedure10 practices here to help solidify this technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/2 mL Syringe, 27 G</td>
			<td>BD</td>
			<td>305620</td>
			<td>https://www.bd.com/en-ca/offerings/capabilities/bd-luer-lok-syringe-with-attached-needle/305620</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Qualoty Biological</td>
			<td>118-156-101CS</td>
			<td>https://www.qualitybiological.com/product/ack-lysing-buffer/</td>
		</tr>
		<tr>
			<td>Biological Irradiator</td>
			<td>Kimtron</td>
			<td>IC-250</td>
			<td>https://www.kimtron.com/ic-250</td>
		</tr>
		<tr>
			<td>Chlorhexidine</td>
			<td>Nolvasan</td>
			<td>NDC 54771-8701-1</td>
			<td>https://www.zoetisus.com/products/petcare/nolvasan-skin-and-wound-cleanser</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, proof 190</td>
			<td>Gold Shield</td>
			<td></td>
			<td>https://goldsd.com/line-card/</td>
		</tr>
		<tr>
			<td>FACSCanto II&#160;</td>
			<td>(Becton Dickinson and Company (BD), Franklin Lakes, NJ, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Omega Scientific</td>
			<td>FB-01</td>
			<td>https://www.omegascientific.com/product/fetal-bovine-serum-usda-certified/</td>
		</tr>
		<tr>
			<td>Flow cytometer, AriaII</td>
			<td>Beckton Dickinson (BD)</td>
			<td></td>
			<td>cell sorting</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td>https://www.thermofisher.com/order/catalog/product/12440053</td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Dechra</td>
			<td>NDC 17033-094-25</td>
			<td>https://www.dechra-us.com/our-products/us/companion-animal/dog/prescription/isoflurane-usp-inhalation-anesthetic</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice&#160;</td>
			<td>Jackson Laboratory, Bar Harbor, ME, USA</td>
			<td> 
			Strain #:005557</td>
			<td>Six- to ten-week-old male or female&#160;</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment, USP</td>
			<td>Bausch Lomb</td>
			<td>NDC 24208-780-55</td>
			<td>https://www.bausch.com/contentassets/2914df881e4344a 
			7a202cc5a0673c977/neomycin-and-polymyxin-b-sulfates-gramicidin-ophthalmic-solution.pdf</td>
		</tr>
		<tr>
			<td>OstiFen Injection (Caprofen)</td>
			<td>VetOne</td>
			<td>NDC 13985-748-20</td>
			<td>http://vetone.net/Default/CatHeaderPage?id=9534ccca-eac5-4d68-8ad8-46436067587b</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Betadine</td>
			<td>NDC 67618-155-16</td>
			<td>https://www.betadine.com/veterinary-surgical-scrub-and-solution/</td>
		</tr>
		<tr>
			<td>TokeOni (in the U.S.)</td>
			<td>Sigma-Aldrich</td>
			<td>808350-5MG</td>
			<td>AkaLumine-HCl/Akaluc; https://www.sigmaaldrich.com/US/en/product/aldrich/808350</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td>https://www.thermofisher.com/order/catalog/product/15575020</td>
		</tr>
		<tr>
			<td>Engraftment antibody panel (in vivo, mouse bone marrow)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antigen</td>
			<td></td>
			<td></td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Antigen: Anti-human CD45 
			Fluorophore: V450 
			Clone: HI30</td>
			<td>BD Biosciences</td>
			<td>560367</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>Antigen: Anti-mouse CD45.1 
			Fluorophore: PE-Cy7 
			Clone: A20</td>
			<td>eBioscience</td>
			<td>25-0453-81</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Antigen:Anti-mouse TER-119 
			Fluorophore: PE-Cy5 
			Clone: TER-119</td>
			<td>eBioscience</td>
			<td>15-5921-83</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Antigen:Anti-human CD3 
			Fluorophore: APC-Cy7 
			Clone: SK7</td>
			<td>BD Biosciences</td>
			<td>341090</td>
			<td>1:12.5</td>
		</tr>
		<tr>
			<td>Antigen:Anti-human CD19 
			Fluorophore: APC 
			Clone: HIB19</td>
			<td>BD Biosciences</td>
			<td>555415</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>Antigen:Anti-human CD33 
			Fluorophore: PE 
			Clone: WM53</td>
			<td>BD Biosciences</td>
			<td>555450</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>Live/Dead staining</td>
			<td></td>
			<td></td>
			<td>Conc.</td>
		</tr>
		<tr>
			<td>PI</td>
			<td>Invitrogen</td>
			<td></td>
			<td>1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Compensation beads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Negative control</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>See instruction for details</td>
		</tr>
		<tr>
			<td>Anti-mouse Ig, &#954;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>See instruction for details</td>
		</tr>
	</tbody>
</table>

simplified intrafemoral injections using live mice allow for continuous bone marrow analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

Following these protocols1,14,15,16,17,18,19,20, each sample was transplanted, and the xenograft mouse model was established. The aim of the experiments here was to show IF injection with several types of human cells and following bone marrow aspiration/ana...

Watch this Scientific Journal Video about Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis at JoVE.com

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

1.6K Views.  Stanford University School of Medicine. This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Video: Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.