We would like to thank Alvin Welch and Larry Pang for their technical assistance. This work was funded in part by the National Institutes of Health (NIH) award P50 CA086306, the California Institute for Regenerative Medicine (CIRM) grants RC1-00149-1 and RS1-00203-1; CIRM New Faculty Award RN2-00902-1, the Caltech-UCLA Joint Center for Translational Medicine, UCLA Center for AIDS Research (CFAR) NIH/NIAID AI028697, the UCLA AIDS Institute, the CIRM Tools and Technology Award RT1-01126, and the UC Multicampus Research Program and Initiatives from the California Center for Antiviral Drug Discovery (number MRPI-143226).

A. Generation of BLT mice
The generation of BLT mice is divided into three (3) parts: (1) processing of fetal tissue and preparation of CD34 human hematopoietic stem cells, (2) transplantation of human tissue, and (3) secondary transplant. For all the protocols, we have provided Table 1 with reagent information.
1. Tissue Processing and Isolation of CD34 Human Hematopoietic Stem Cells
Fresh fetal tissue or tissue shipped overnight on ice from various organ procurement agencies can be used. Often fetal tissue is not sterile, as evidenced by the production of 1,000 bacterial colonies on blood agar per milliliter of surrounding medium. We routinely wash the tissue twice in 40 ml of sterile PBS. While this does not remove all bacteria, it can make a difference between a successful transplant series and an outcome in which most of the recipient mice succumb to bacterial infection. As an added step, to further disinfect the tissue, we culture the cell suspension in the presence of antibiotics.
1.1 Processing of Fetal Thymic Tissue
<ol>
	<li>Wash thymus by pouring off the medium into a beaker of bleach and using scalpel to prevent the tissue from sliding into the bleach. Fill up the tube with PBS. Cap and invert a few times to wash the thymus. Decant supernatant into bleach. Repeat.</li>
	<li>Place the thymus tissue in 8 ml of RPMI + 10% Fetal Calf Serum in a 60-mm dish and shear into 1.5 to 2 mm pieces with two scalpels. Shearing with scalpels minimizes tearing and damaging the tissue bits. Damaged tissue tends to become swollen, sticky, and difficult to draw into the implant needle. The number of bits obtained is the first upper limit on transplantable mice.</li>
	<li>Transfer the suspended bits with a 25 ml pipet (to minimize tissue damage) into a T25 flask. Add 70 &mu;l of piptazo (Zosyn) to flask and culture overnight, at 37 &deg;C, 5% CO2. This considerably reduces contaminating bacteria. If you wish to do tissue typing or any other phenotyping assays, take out 1 ml of cells.</li>
</ol>
1.2 Processing of Fetal Liver Tissue
<ol>
	<li>Wash the liver as in step 1.1.1.</li>
	<li>Add 10 ml of Iscove's medium and decant everything into a 100 mm Petri dish.</li>
	<li>With two scalpels, dice the liver into small (~3 mm) pieces. If you run into white connective tissue, scrape the liver from it and discard it in the bleach.</li>
	<li>Homogenize the tissue by sucking it into a 12 ml syringe fitted with a 16 gauge blunt needle 3 or 4 times and transferring to a 50 ml tube.</li>
	<li>Prepare the enzymes for tissue digestion as follows: To 10 ml of Iscove's in a 50 ml tube, add 200 &mu;l of each: collagenase type IV (1 mg/ml), hyaluronidase (1 mg/ml), DNase I (2 U/ml).</li>
	<li>Draw enzymes into a 12 ml syringe and fit it with a 0.22 &mu; filter. Filter the enzyme/medium directly into the cell suspension.</li>
	<li>Cap and seal the cells with Parafilm. Rotate at 37 &deg;C for 90 min.</li>
	<li>Set up a 50 ml tube with a 100 &mu; cell strainer. Pipet the cell digest through this strainer.</li>
	<li>Add PBS to the cells to bring the volume up to 50 ml. Split this into two tubes of 25 ml.</li>
	<li>Underlay the cells with 10 ml of Ficoll. Spin at 2,400 rpm for 20 min without brake.</li>
	<li>The interface should be thick. Suck it out with a 5 ml pipet and transfer to another tube. You should have two tubes of interface. Add 50 ml of PBS to each and spin at 1,200 rpm for 10 min.</li>
	<li>Combine pellets into one tube and wash three more times with PBS containing 2% FCS.</li>
	<li>Finally suspend in 50 ml of RPMI + 10% FCS and count cells using trypan blue. Depending on the size of the tissue, you can get from 108 to 109 total hepatocytes.</li>
	<li>Transfer the cells to a T150 flask and add 30 ml of RPMI + 10% FCS and 0.8 ml of piptazo.</li>
	<li>Incubate for 1 to 1.5 hr at 37 &deg;C to remove adherent cells.</li>
</ol>
1.3 Sorting and Transduction of CD34 Hematopoietic Stem Cells
<ol>
	<li>After attachment of cells in step 1.2.15, agitate the flask back and forth gently for a minute to dislodge loose cells. There will be a lot of fibroblasts and such stuck to the surface.</li>
	<li>Count again using a 1 to 20 dilution and record the total number of cells. Save a few tenths of a ml for later staining. You should have approximately 30% fewer cells from your initial cell counts (step 1.2.13).</li>
	<li>Spin down and wash two times with 40 ml of MACS buffer.</li>
	<li>CD34 cells are sorted using Miltenyi Biotech's CD34 Direct Kit. We follow exactly the manufacturer's protocol using LS columns provided by the same company.</li>
	<li>Recovered cells will be in 5 ml. Count and calculate total cells. Your yield of CD34+ cells should be 3-5% of your total cell counts. This varies with the age of the fetal liver. Younger fetal livers tend to give a higher yield of CD34+ cells.</li>
	<li>Divide the number of cells by 1x106. This gives the second upper limit on transplantable mice.</li>
	<li>Count the CD34- fraction (flow through and washes) and reserve 6 x 106per mouse to be transplanted. Culture these cells overnight in 50 to 80 ml of RPMI + 10% Fetal Calf Serum + 1% piptazo.</li>
	<li>Transduce the CD34+ cells overnight with your lentiviral vector. We use retronectin by Takara following exactly the manufacturer's instructions.</li>
	<li>The next day, harvest the CD34- cells, the transfected CD34+ cells and count.</li>
	<li>Split the CD34+ cells in half: Part A and Part B.</li>
	<li>Spin down Part A CD34+ transduced cells and suspend in 4 to 6 ml of freezing medium (RPMI + 10% FCS + 10% DMSO, 0.22 &mu; sterile filtered). Put 1 ml aliquots into freezing vials and label with "huFetalCD34", the number of cells in the vial, the date, and your initials.</li>
	<li>For each mouse mix 0.5x106Part B CD34+ transduced cells and 4.5x106CD34- cells in a sterile screw-cap microfuge tube, pellet and keep on ice. Also thaw a tube of Matrigel (BD Biosciences) on ice. All these tubes must be kept on ice until used (go to step 2.10).</li>
</ol>
2. Tissue Transplantation
The goal of this step is to transplant a human fetal thymus/liver organoid under the NSG mouse kidney capsule. This organoid better mimics the process of human T cell selection and maturation processes as the human hematopoietic stem cells will use the human thymus and not the mouse as the site for their differentiation to different T cell and other lymphoid lineages. The use of CD34- cells in the transplantation process is to re-generate the fetal liver stroma and allowing for better transplantation and growth of the implant. The age of NSG mice used is 6-8 weeks old.
<ol>
	<li>Preparation of drugs for surgery:
	<ol class="lalpha">
		<li>Isoflurane: Roll up a 2 inch gauze pad and stuff it into a 15 ml screwcap centrifuge tube. Pour in about 1 ml of isoflurane directly from the bottle.</li>
		<li>Ketamine/zylazine: Add 0.52 ml of ketamine (Ketaset) (100 mg/ml) and 0.52 ml of xylazine (Anased) (100 mg/ml) to a 20 ml vial of saline for injection. Label with the expiration date of the earliest expiring component and keep frozen at -20 &deg;C until needed.</li>
		<li>Carprofen (Rimadyl). Dilute this just before a surgical procedure. Dilute 1:100 by withdrawing 0.1 or 0.2 ml from the vial with a 1 ml syringe and a 25G needle. Inject into a 10 or 20 ml of saline for injection. Label and date. Keep in the fridge for one day only. Load into 3 ml syringes with 25G needles.</li>
	</ol>
	</li>
	<li>Set up the table for surgeries. Cover it with sterile blue pads, and a sterile towel for each surgeon. Each surgeon gets from their box of sterilized instruments: 4" scissors, curved blunt forceps, needle-nose forceps, hemostat, 16G blunted trochar, and wound clip applicator. In addition, they need a curved-needle Vicryl suture, a stack of isopropanol wipe packets, a 35 mm Petri dish of PBS, and a 1 ml syringe filled with PBS.</li>
	<li>Set in the middle of the table a glass Coplin jar with 2 cm of Betadine and 2 cotton swabs. Pour the thymus bits into a 60 mm Petri dish.</li>
	<li>The anesthesia is performed on a separate table or in a hood covered with sterile blue pads. Set up two small racks and an electronic balance with a beaker to hold the mice. Use one rack to hold 0.5 ml X 28G insulin syringes loaded with ketamine/xylazine and the other to hold the 3 ml syringes of carprofen. Also move the biohazard waste container near to hold the shaved fur.</li>
	<li>Weigh all the mice (6-8 week old) in a cage to get the average weight and load syringes with a dose of 15 &mu;l/g of ketamine/xylazine. Inject all the mice in the cage.</li>
	<li>After the mice become sluggish shave their left side from hip to shoulder between the center of the back and the belly with an Oster clipper with No. 40 blade. Record their weight, and punch their ears for numbering.</li>
	<li>Inject 0.3 ml carprofen subcutaneously over the shoulder.</li>
	<li>Check the anesthesia level of the mouse by squeezing a paw. If the mouse flinches, administer isoflurane from a tube for about 12 sec. Then try the paw squeeze again. Keep giving short shots of isoflurane until the paw reflex disappears. Lay the mouse on its right side facing left.</li>
	<li>A 16-gauge cancer implant needle, with the tip filed round is used to insert the tissue. Flush out the trochar a few times in the dish of PBS. Holding it horizontal with the rod just inside the tip. Add a piece of thymus with the needlenose forceps and suck it in.</li>
	<li>A helper adds 5 &mu;l of cold Matrigel to one tube of cells (step 1.3.12) with an Eppendorf positive displacement pipet, and mixes by gentle stirring. The surgeon holds the trochar horizontally and pulls the rod back slowly as the helper pipets the Matrigel mix into the trochar. The mix gels at room temperature.</li>
	<li>Swab the bare skin with Betadine. Then wipe this off with an isopropanol wipe. Repeat.</li>
	<li>Locate the spleen under the skin. It is the darkest spot. The mouse's kidney is located about 5 mm dorsal to the spleen, which can be seen through the shaved skin.</li>
	<li>Pick up the skin over this spot with the blunt forceps and make a cut parallel to the spleen about 15 mm long. Make a similar cut in the peritoneum muscle layer below. In males, the kidney should be directly visible and you simply have to squeeze the abdomen to pop it out. In females, you may have to pick up the ovary with the hemostat and drag out the kidney. This may help retain the kidney outside of the body. For males, you will have to keep some pressure on the abdomen with your left hand to keep the kidney exposed.</li>
	<li>Pluck a little hole in the posterior end of the kidney capsule. It may bleed a little.</li>
	<li>Slide the trochar into this hole and along the kidney until the orifice of the trochar is completely covered by the kidney capsule. Then using the little finger of your right hand, inject the tissue.</li>
	<li>Push the kidney back in gently with the closed hemostat. Put one stitch in the peritoneum tied with a double know. Squeeze the skin up like a purse and put in two wound clips.</li>
	<li>Put a drop of PBS on each eye and lay the mouse on its side on the cage bedding. Make sure all the mice have returned to laying on their bellies before leaving them.</li>
	<li>Next day, give each mouse another shot of carprofen.</li>
</ol>
3. Secondary Transplantation
The goal of this step in the generation of BLT mice is to populate the moue bone marrow with human hematopoietic stem cells. The transplanted implant from procedure (2) is not sufficient to support full reconstitution of the human immune system in these mice. During the secondary transplant, we sub-lethally irradiate the mice to deplete murine bone marrow cells thus generating "space" for the implantation of the human CD34+ cells.
<ol>
	<li>Four to six weeks later irradiate the mice with 2.7 Gy. The mice are injected the same day with Part A CD34+ transduced cells. The timeframe is based on the establishment and growth of the thy/liv implant transplanted in step 2. Typically, during this time the implant has grown significantly allowing us to proceed to the next steps.</li>
	<li>Thaw out the Part A CD34+ transduced cells, wash and suspend in medium to a concentration of 5x106/ ml.</li>
	<li>Load 0.5 ml X 28G insulin syringes with 0.1 ml of cells (105 to 5x105 cells per 0.1 ml per mouse) for one cage of mice (each cage contains a maximum of 5 mice) and lay them on a rack facing away.</li>
	<li>Anesthetize a mouse with an isoflurane tube by scruffing it and holding its nose in the tubes for about 20 sec. Count to keep track of the time.</li>
	<li>Then face it to the right, and pull the skin around its neck tight with the thumb and forefinger of the left hand. Press under its jaw with your third finder so that its right eye bulges out. Holding the syringe parallel to the long axis of the mouse's head, slip the needle behind the eye and slide it gently until it stops. Don't press any further, but inject the load over about 2 sec.</li>
	<li>Two to three months later mice are bled retroorbitally using EDTA coated glass pipettes. You can only take a maximum of 200 &mu;l of blood per month per mouse. 50-100 &mu;l of blood should be sufficient for FACS analysis. Alternative bleeding strategies include facial vein bleeds and tail vein bleeds. The latter will give you very small volumes of blood that may not be sufficient for multiple assays.</li>
	<li>Blood samples are stained for the expression of the human lymphocyte marker CD45 to assess reconstitution. Blood samples are added into 1 ml red blood cell lysis buffer (160 mM NH4Cl, 100 mM KHCO3, 0.01 mM EDTA) and incubated for 5 min at room temperature.</li>
	<li>Wash the cells in lysis buffer, 5 min at 1,200 rpm, and repeat if necessary.</li>
	<li>Centrifuge cells as in 3.8 and wash in PBS supplemented with 3% fetal calf serum (FACS-PBS).</li>
	<li>Remove supernatant and re-suspend pellets in 100 &mu;l FACS-PBS.</li>
	<li>Add antibodies to assess human lymphocyte reconstitution. We typically include the following CD45 (human lymphocyte marker), CD8, CD4, CD14 and CD16 for macrophages and monocytes, CD19 for B cells and CD56 for NK cells.</li>
	<li>Incubate at 4 &deg;C for 30 min.</li>
	<li>Wash as in step 3.9 and re-suspend cells in 2% paraformaldehyde for FACS analysis.</li>
	<li>If mice are reconstituted, we proceed with tumor injections. Reconstitution levels of human lymphocytes vary, from less than 1% to 40% of total blood cells. The levels and types of lymphocyte lineages are comparable to what one would expect from healthy human blood donors. However, do expect fluctuations in the number of non-T cell lineages. If the numbers are not acceptable within the 12-week timeframe you could repeat the secondary transplant procedure from step 3.1.</li>
	<li>Tumor cell lines are cultured according to manufacturer's or laboratory recommendations. Cells are harvested, washed and resuspended in matrigel at a 1:1 ratio (50 &mu;l cells/50 &mu;l matrigel). Samples are kept on ice at all times.</li>
	<li>Mice are anesthetized and shaved at the area of injection. The area is sterilized using an isopropanol pad. A helper loads cell suspensions in pre cooled 23G 1 ml syringes. Tumor cells are injected subcutaneously. Treat the site of injection with antibiotic ointment. Tumors should establish and begin growing in 4 weeks.</li>
</ol>
B. In vivo Positron Emission Tomography (PET)
For our studies we examine glucose uptake ([18F]-fluorodeoxyglucose ([18F]FDG) thus mice are fasted 4-6 hr prior to imaging. MicroPET/CT scans are done using the microPET Inveon scanner (Siemens Preclinical Solutions) and MicroCATII CT scanner (Siemens PreclinicalSolutions). Image analysis is done using OsiriX (Pixmeo, Switzerland) software. The goal is to measure the metabolic activity of the tumor and ultimately to use PET imaging as an alternative to physically measuring tumor regression. As seen in Figure 2, we have encountered tumors that based on physical appearance and size are not targeted but live PET imaging revealed extensive tissue necrosis. This methodology can serve as a more sensitive and accurate indicator of tumor regression.
<ol>
	<li>Designate one chamber for anesthetizing (2% isofluorane) the mice, and one chamber as the 60 min uptake of FDG before PET scanning ("waiting chamber"). Place blue pads in both chambers.</li>
	<li>As animals are anesthetized over a long periods, they should be kept warm at 30 &deg;C through the use of water filled gloves that are warmed in a microwave and placing the cages on heat pads.</li>
	<li>Turn the isoflurane flow for this chamber on. Allow the isoflurane to fill the anesthetizing chamber for approximately 5-10 min before placing any mice into the chamber. Make sure the lid is locked and closed tightly to avoid leaking isoflurane.</li>
	<li>Place the first mouse into the anesthetizing chamber and lock the lid tightly. Allow 5-10 min or until the mouse is unconscious.</li>
	<li>Remove the mouse from the anesthetizing chamber, label the tail of the mouse (different color per cage) and inject intraperitoneally the mouse with [18F]FDG (80 &mu;Ci). Take note of the time the mouse was injected. The mouse will be anesthetized again 50 min from this timepoint, allowing for 10 min to set up for PET scanning (for the total of 60 min [18F]FDG uptake before PET scanning).</li>
	<li>Place the injected mouse into the waiting chamber, allowing [18F]FDG uptake for 1 hr before PET scanning. Do not lock the lid. Leave the lid loose to allow airflow.</li>
	<li>Immediately place the next mouse for FDG injection into the anesthetizing chamber. Subsequent mice injections should be spaced 10 min apart, because the process of PET scanning is 10 min. Immediately after one mouse is scanned, the 60 min FDG uptake for the next mouse to be scanned will be ready exactly 10 min apart.</li>
	<li>Continue anesthetizing and injecting mice as stated in steps 7-9.</li>
	<li>After 50 min from the first mouse injection timepoint, place the first injected mouse into the anesthetizing chamber again (while still continuing to anesthetize and inject mice).</li>
	<li>Connect the mouse bed to the oxygen and isoflurane lines, and turn the isoflurane flow on for the bed. Place a blue pad on the bed.</li>
	<li>Place the mouse ready for PET scanning onto the bed, stretching the arms and legs and holding this position with tape. Place lubricant over the eyes. This step is the most crucial since the positioning of the animal can affect the quality of your imaging.</li>
	<li>Disconnect the isoflurane and oxygen lines, and quickly mount the bed on the PET scanning machine. Place the isoflurane and oxygen lines. Make sure the isoflurane flow is on for the PET scanning machine. Connect the PET scanning cable and begin the scan.</li>
	<li>Once the PET scan is complete then move the animal to the CT scanner. Following this scan move the animal to its respective cage.</li>
</ol>

<ol>
	<li>McCune, J. M., Namikawa, R., Kaneshima, H., Shultz, L. D., Lieberman, M., 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=The+SCID-hu+mouse:+murine+model+for+the+analysis+of+human+hematolymphoid+differentiation+and+function.">The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function.</a> Science. 241 (4873), 1632-1639 (1988).</li><li>Bristol, G. C., Gao, L. Y., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+maintenance+of+SCID-hu+mice+for+HIV+research.">Preparation and maintenance of SCID-hu mice for HIV research.</a> Methods. 12 (4), 343-347 (1997).</li><li>Mosier, D. E., Gulizia, R. J., Baird, S. M., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+a+functional+human+immune+system+to+mice+with+severe+combined+immunodeficiency.">Transfer of a functional human immune system to mice with severe combined immunodeficiency.</a> Nature. 335 (6187), 256-259 (1988).</li><li>Melkus, M. W., Estes, J. D., Padgett-Thomas, A., Gatlin, J., Denton, P. W., Othieno, F. A., Wege, A. K., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1.">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nat. Med. 12 (11), 1316-1322 (2006).</li><li>Denton, P. W., Estes, J. D., Sun, Z., Othieno, F. A., Wei, B. L., Wege, A. K., Powell, D. A., Payne, D., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiretroviral+pre-exposure+prophylaxis+prevents+vaginal+transmission+of+HIV-1+in+humanized+BLT+mice.">Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.</a> PLoS Med. 5 (1), e16 (2008).</li><li>Sun, Z., Denton, P. W., Estes, J. D., Othieno, F. A., Wei, B. L., Wege, A. K., Melkus, M. W., Padgett-Thomas, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarectal+transmission,+systemic+infection,+and+CD4++T+cell+depletion+in+humanized+mice+infected+with+HIV-1.">Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.</a> J. Exp. Med. 204 (4), 705-714 (2007).</li><li>Shimizu, S., Hong, P., Arumugam, B., Pokomo, L., Boyer, J., Koizumi, N., Kittipongdaja, P., Chen, A., Bristol, G., Galic, Z., Zack, J. A., Yang, O., Chen, I. S., Lee, B., An, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+short+hairpin+RNA+potently+down-regulates+CCR5+expression+in+systemic+lymphoid+organs+in+the+hu-BLT+mouse+model.">A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.</a> Blood. 115 (8), 1534-1544 (2010).</li><li>V, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+HIV+Latency+in+Humanized+BLT+Mice.">Generation of HIV Latency in Humanized BLT Mice.</a> J. Virol. 86 (1), 630-634 (2012).</li><li>Vatakis, D. N., Koya, R. C., Nixon, C. C., Wei, L., Kim, S. G., Avancena, P., Bristol, G., Baltimore, D., Kohn, D. B., Ribas, A., Radu, C. G., Galic, Z., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+from+antigen-specific+CD8+T+cells+generated+in+vivo+from+genetically+engineered+human+hematopoietic+stem+cells.">Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (51), 1408-1416 (2011).</li></ol>

No conflicts of interest declared.

The modified BLT humanized mouse model coupled with in vivo PET imaging are powerful tools to study chronic human diseases. This system takes the BLT mouse model and advances it beyond the limited scope for HIV research. In addition, it is a great system in which we can examine various gene therapy protocols as well as diagnostic techniques before they can reach the clinical setting. The latter coupled with the low cost of using mice versus primates makes this a very useful model.
<p class="jove_conten...

<table border="1">
 <tbody>
 <tr>
 <td>Name 				of the reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Comments 				(optional)</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td>10010049</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI 				1640</td>
 <td></td>
 <td>A1049101</td>
 <td></td>
 </tr>
 <tr>
 <td>Iscove's</td>
 <td></td>
 <td>12440061</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal 				Calf Serum</td>
 <td></td>
 <td>16000085</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase 				type IV</td>
 <td>Sigma 				Aldrich</td>
 <td>C5138</td>
 <td></td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td></td>
 <td>H3506</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse 				I</td>
 <td>Roche 				Diagnostics</td>
 <td>10104159001</td>
 <td></td>
 </tr>
 <tr>
 <td>Piptazo 				(Zosyn)</td>
 <td>Pfizer</td>
 <td></td>
 <td><a href="http://labeling.pfizer.com/showlabeling.aspx?id=416" target="_blank"> Piperacillin/tazobactam 				combination </a></td>
 </tr>
 <tr>
 <td>Ficoll 				(Ficoll-Paque Plus)</td>
 <td>Ge 				Healthcare Life Sciences</td>
 <td>17-1440-02</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS 				Buffer</td>
 <td>Miltenyi 				Biotech</td>
 <td>See 				comments</td>
 <td>MACS 				buffer: PBS supplemented with 0.5% fetal bovine serum and 2 mM 				citrate dextrose formula-A </td>
 </tr>
 <tr>
 <td>CD34 				Microbead Kit</td>
 <td></td>
 <td>130-046-702</td>
 <td></td>
 </tr>
 <tr>
 <td>LS 				Columns</td>
 <td></td>
 <td>130-042-401</td>
 <td></td>
 </tr>
 <tr>
 <td>Retronectin</td>
 <td>Takara</td>
 <td>T100A</td>
 <td></td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>BD 				Biosciences</td>
 <td>354263</td>
 <td></td>
 </tr>
 <tr>
 <td>Isofluorane</td>
 <td>Baxter</td>
 <td></td>
 <td><a href="http://www.baxter.com/healthcare_professionals/products/forane.html" target="_blank">http://www.baxter.com/healthcare_professionals/products/forane.html</a></td>
 </tr>
 <tr>
 <td>Carprofen 				(Rimadyl)</td>
 <td>Pfizer 				Animal Health</td>
 <td></td>
 <td><a href="https://www.rimadyl.com/default.aspx" target="_blank">https://www.rimadyl.com/default.aspx</a></td>
 </tr>
 </tbody>
</table>

using the blt humanized mouse as a stem cell based gene therapy tumor model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

A flow chart of the transplantation process is shown in Figure 1A. A picture of the thy/liv implant is shown in Figure 1B. The thymic tissue develops normally and has a physiological distribution of human CD4 and CD8 T cells. Following reconstitution, the animals carry a human immune system with normal distribution of CD4, CD8 T cells and other immune cell lineages.
The discrepancy between tumor size and live tissue is shown in Figure 2. ...

Watch this Scientific Journal Video about Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model at JoVE.com

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

using-blt-humanized-mouse-as-stem-cell-based-gene-therapy-tumor

Research

JoVE Journal

Immunology and Infection

18.6K Görüntüleme.  David Geffen School of Medicine at UCLA. Hümanize fareler kullanılarak tümör spesifik T hücrelerin oluşturulması ve karakterizasyonu burada tanımlanmıştır. İnsan timik doku ve genetiği değiştirilmiş insan hematopoietik kök hücrelerin bağışıklığı baskılanmış farelere nakledilmektedir. Anti-tümör bağışıklık yanıtı in vivo incelenmesi için izin veren bir mühendislik insan bağışıklık sisteminin sulandırma ile sonuçlanır.

Video: Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...