This work was funded in part by sponsored research agreements from TRex Bio and grants from the NIH (1R01AR075864-01A1). JMM is supported by the Cancer Research Society (grant 26005). We acknowledge the Parnassus Flow Cytometry Core supported in part by grants NIH P30 DK063720, S10 1S10OD021822-01, and S10 1S10OD018040-01.

The present study was approved and performed in compliance with UCSF IACUC (AN191105-01H) and IRB (13-11307) protocols. Skin samples, discarded as part of routine elective surgical procedures, such as hernia repair, were used for the present research. The skin samples are either de-identified and certified as Not Human Subjects Research or, if clinically identifying information is required for downstream analyses, patients provided written consent under IRB protocol 13-11307. No other inclusion or exclusion criteria were utilized. NSG mice of either sex, 8-10 weeks of age, were employed in the study. The mice were obtained from commercial sources (see Table of Materials).
1. Processing of donor human skin sample
NOTE: The human skin sample used in this transplant was a large sample collected from the abdomen of a healthy patient. The sample must be at least 15 cm x 7.5 cm. Size limitations may affect the number of mice for which skin is available and the choice of graft size.
<ol>
	<li>Maintain the skin sample at cold temperatures (on ice; 4&#176;C) before preparation and grafting. Keep the sample moist in a closed specimen collection cup with the gauze soaked in phosphate-buffered saline (PBS). 
	NOTE: Storing the skin sample at 4 &#176;C for longer than 2 days is not recommended. However, reports exist where the skin samples are stored for a longer time12. 
	CAUTION: Treat all human tissue with standard biohazard precautions.</li>
	<li>Prepare to dermatome the human skin sample in a sterilized negative pressure tissue culture hood on a sterilized dissection board.</li>
	<li>Place the skin sample, epidermis side up, on the dissection board. Wipe the epidermis with a sterile alcohol prep pad and then with PBS.</li>
	<li>Pin the closer edge of the skin in place with a 1.5 in dissecting T-pin (see Table of Materials).</li>
	<li>Dermatome the skin specimen at a 400 &#181;m thickness, applying steady pressure while cutting forward at a 30&#176;-45&#176; angle. Follow all instrument-specific instructions and safety measures (see Table of Materials). 
	NOTE: For details on the dermatome technique, please see a previously published report13.</li>
	<li>Prepare a 100 mm x 20 mm Petri dish by placing a sterile gauze soaked in sterile PBS at the bottom of the dish. Place the skin, epidermis side up, onto the wet gauze.</li>
	<li>Seal and cover the plate edges with a semitransparent sealing film (see Table of Materials) to ensure the sample is not contaminated. Store the sample at 4&#176; C prior to grafting.</li>
</ol>
2. Pre-surgery conditioning and preparation
<ol>
	<li>Prepare the sterile instruments and a sterile surgical station for grafting. Use autoclaved paper towels as sterile surfaces for instrument and mouse placement. 
	NOTE: Mice may be grafted after weaning but are preferably grafted between 8-10 weeks of age. Mice of either sex may be grafted.</li>
	<li>Perform the surgical preparation, such as hair removal, in an area physically separated from the surgical station.</li>
	<li>Prepare the anti-GR1 (see Table of Materials) by diluting it to 1 mg/mL in sterile saline. Dose each mouse with 100 &#181;g/100 &#181;L of the anti-GR1 solution intraperitoneally following anesthesia induction.</li>
	<li>Anesthetize the mice, one at a time, with isoflurane or other institutionally approved anesthetics. 
	NOTE: Isoflurane needs to be given at a 3%-5% concentration during induction. Once the mouse is immobile, lower the isoflurane concentration to 1%-3% to effect for the duration of the surgery.
	<ol>
		<li>Monitor the mouse for appropriate depth of anesthesia by observing respiratory rate, absence of toe-pinch response, and appropriate pink coloration of ears and mouth. 
		CAUTION: Use appropriate anesthetic machinery and scavenging methods, and avoid exposure to isoflurane vapors.</li>
	</ol>
	</li>
	<li>Transfer the mouse to a heating pad or another heat source (see Table of Materials).</li>
	<li>Administer the ophthalmic ointment by dabbing a small drop of ointment on the eye with a gloved finger.</li>
	<li>Administer analgesics Buprenorphine (0.08 mg/kg) and Carprofen (5 mg/kg) (see Table of Materials) subcutaneously by pinching the skin and injecting at an angle parallel to the body. 
	NOTE: Prepare the pre-treatment analgesia following institutional protocols. Follow institutional guidelines for the selection and administration of analgesics. The method of analgesia used in this study is outlined in step 2.7 and Supplementary Figure 1.</li>
	<li>Administer the anti-GR1 (prepared in step 2.4) intraperitoneally by slightly lifting the mouse by the tail, exposing the abdomen, and injecting at a 30&#176; angle using an insulin 1 mL (12.7 mm) syringe.</li>
	<li>Use animal-safe electric clippers (see Table of Materials) to shave the middle and upper portions of the dorsal side of the mouse.</li>
	<li>Clear all the hair and apply a generous amount of hair removal ointment onto the shaved skin for 30 s to 1 min.</li>
	<li>Completely wipe away hair removal ointment with a paper towel and PBS.</li>
</ol>
3. Transplantation procedure
<ol>
	<li>Transfer the mouse to a secondary surgical location, away from the hair removal station.</li>
	<li>Sterilize the surgical site with the iodine swab stick in a circular motion, starting in the middle and working out toward the edge of the depilated area.</li>
	<li>Place a piece of sterile plastic wrap over the mouse and cut a window in the plastic slightly larger than the size of the area to be grafted.</li>
	<li>Cut a rectangle-shaped 10 mm x 10 mm portion of donor skin to be grafted with a scalpel. Do this by firmly holding the donor skin in place with the backside of the forceps and cutting alongside the forceps with the scalpel.</li>
	<li>Using the surgical scissors, snip a rectangular area of mouse skin matching the size of the donor skin piece, creating a graft bed. Use&#160;forceps to pull the skin away from the body, and cut the skin with the scissors angled away from the body to avoid cutting deeply into the facia.</li>
	<li>Place the donor skin piece, epidermis side up, onto the prepared graft bed.</li>
	<li>Using the back of the forceps, manipulate the skin, sliding back and forth until the donor skin lies completely flat against the graft bed.</li>
	<li>Add drops of surgical glue tissue adhesive (see Table of Materials) where the donor skin meets the mouse skin and hold the mouse and donor skin together with forceps for 1-2 s so that the glue adheres to the tissues. Completely seal the edge of the graft and allow for the glue to dry fully.</li>
	<li>Bandage the mice (Figure 1) following the steps below.
	<ol>
		<li>Cut a piece of petrolatum gauze (see Table of Materials) large enough to cover the graft area completely.</li>
		<li>Cover the graft with the petrolatum gauze, and lightly press the gauze against the skin using forceps.</li>
		<li>Cut a strip of a transparent film dressing lengthwise so that the width is large enough to cover the mouse&#39;s wound.</li>
		<li>Firmly press the transparent film dressing, adhesive side down, over the gauze. Quickly roll the mouse to wrap the dressing completely around the torso, ensuring it fits tightly without impeding respiration and all limbs are free for movement.</li>
		<li>Place the mouse in a recovery cage and monitor it until it is alert and moving around. Provide a heat source on part of the cage for at least 15 min following recovery. 
		NOTE: The animals are expected to recover within 1-5 min after placing them in the recovery cage.</li>
	</ol>
	</li>
	<li>Singly house the mice following grafting.</li>
	<li>Administer post-surgical analgesia as required by institutional protocols. 
	&#8203;NOTE: Buprenorphine (0.08 mg/kg) was administered subcutaneously 4-6 h later during the present study.</li>
</ol>
4. Post-surgical procedures
<ol>
	<li>Inject 100 &#181;L (100 &#181;g) of anti-GR1 intra-peritoneally at 4 days, 7 days, and 11 days post-grafting to prevent graft rejection (Supplementary Figure 1).</li>
	<li>Replace bandages as necessary and if removed by mice.</li>
	<li>Rebandage all the mice on day 7.</li>
	<li>Remove the bandages on day 14.</li>
	<li>Monitor the mice for signs of graft rejection and systemic inflammation (weight loss, hair loss, extreme lethargy).</li>
	<li>Harvest the mice between 3 and 6 weeks post-graft.
	<ol>
		<li>Euthanize the mice via regulator-controlled carbon dioxide (CO2) administration followed by cervical dislocation. 
		&#8203;NOTE: Follow institutional guidelines for euthanasia.</li>
		<li>Dissect the skin grafts from the mice14. Place a portion of the graft in 10% formalin for 24 h before paraffin embedding and sectioning for histology staining9.</li>
		<li>Mince the skin grafts with scissors and digest enzymatically with 250 IU/mL of Collagenase IV and 0.02 mg/mL of DNase in cell culture media overnight. Stain the cells for surface and intracellular markers following the previously described procedure14.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zomer, H. D., Trentin, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+wound+healing+in+humans+and+mice:+Challenges+in+translational+research.">Skin wound healing in humans and mice: Challenges in translational research.</a> Journal of Dermatological Science. 90 (1), 3-12 (2018).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 969618 (2011).</li><li>Mestas, J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> The Journal of Immunology. 172 (5), 2731-2738 (2004).</li><li>Pasparakis, M., Haase, I., Nestle, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+regulating+skin+immunity+and+inflammation.">Mechanisms regulating skin immunity and inflammation.</a> Nature Reviews Immunology. 14 (5), 289-301 (2014).</li><li>Sun, H., Zhang, Y. -. X., Li, Y. -. M. <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+skin+organoids:+potential+opportunities+and+challenges.">Generation of skin organoids: potential opportunities and challenges.</a> Frontiers in Cell and Developmental Biology. 9, 3176 (2021).</li><li>Crist&#243;bal, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+human+skin+transplantation:+a+systematic+review.">Mouse models for human skin transplantation: a systematic review.</a> Cells Tissues Organs. 210 (4), 250-259 (2021).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nature Reviews Genetics. 19 (11), 671-687 (2018).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD/SCID/&#947;cnull+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/&#947;cnull mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Meraz, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+patient-derived+xenograft+humanized+mouse+model+for+evaluation+of+lung+cancer+immune+responses.">An improved patient-derived xenograft humanized mouse model for evaluation of lung cancer immune responses.</a> Cancer Immunology Research. 7 (8), 1267-1279 (2019).</li><li>Racki, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD-scid+IL2rgamma(null)+mouse+model+of+human+skin+transplantation+and+allograft+rejection.">NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.</a> Transplantation. 89 (5), 527-536 (2010).</li><li>Meehan, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+xenograft+model+of+human+vasculature+in+the+mouse+ear+pinna.">Developing a xenograft model of human vasculature in the mouse ear pinna.</a> Scientific Reports. 10 (1), 2058 (2020).</li><li>Gokkaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+graft+storage+in+platelet+rich+plasma+(PRP).">Skin graft storage in platelet rich plasma (PRP).</a> Dermatologic Therapy. 33 (1), 13178 (2020).</li><li>. The Humeca D42 and D80 battery operated cordless dermatomes Available from: <a target="_blank" href="https://www.youtube.com/watch?v=YCRowX-TdA">https://www.youtube.com/watch?v=YCRowX-TdA</a> (2021)</li><li>Rodriguez, R. 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=Memory+regulatory+T+cells+reside+in+human+skin.">Memory regulatory T cells reside in human skin.</a> The Journal of Clinical Investigation. 124 (3), 1027-1036 (2014).</li><li>Hoogstraten-Miller, S. L., Brown, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+in+rodent+aseptic+surgery.">Techniques in rodent aseptic surgery.</a> Current Protocols in Immunology. 82 (1), 12-14 (2008).</li><li>Karim, A. 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=Evolution+of+ischemia+and+neovascularization+in+a+murine+model+of+full+thickness+human+wound+healing.">Evolution of ischemia and neovascularization in a murine model of full thickness human wound healing.</a> Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. 28 (6), 812-822 (2020).</li><li>Ali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+graft-versus-host-disease+in+NOD-scid+IL-2R&#947;null+mice+display+a+T-effector+memory+phenotype.">Xenogeneic graft-versus-host-disease in NOD-scid IL-2R&#947;null mice display a T-effector memory phenotype.</a> PloS One. 7 (8), 44219 (2012).</li><li>Souci, L., Denesvre, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+skin+models+in+domestic+animals.">3D skin models in domestic animals.</a> Veterinary Research. 52 (1), 21 (2021).</li><li>Holtkamp, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks+guide+dendritic+cells+into+skin+lymphatics.">Circadian clocks guide dendritic cells into skin lymphatics.</a> Nature Immunology. 22 (11), 1375-1381 (2021).</li></ol>

MDR is a founder of TRex Bio and Sitryx. MDR and MML receive research funding from Sitryx, Q32, and TRex Bio.

The mouse xenograft skin transplant model is a key technique to mechanistically dissect human skin immune responses in an in vivo setting14. Successful skin xenograft transplants rely upon appropriate preparation of mice and&#160;skin specimens and mice and adherence to aseptic rodent surgery methods15. Rapid cooling and proper storage of skin samples at cold temperatures in media (such as sterile saline) are important to ensure continued tissue health prior to tra...

Mouse models are frequently used to make inferences about human biology and disease, partly due to their experimental reproducibility and capacity for genetic manipulation. However, mouse physiology does not completely recapitulate human organ systems, particularly skin, and therefore has limitations for use as a preclinical model in drug development1. Anatomical differences between mouse and human skin include differences in epithelial thicknesses and architecture, lack of murine eccrine sweat glands, and variations in hair cycling2. Furthermore, both the innate and adaptive arms of the immune system are divergent between the two species3. Mouse skin contains a unique immune population of dendritic epidermal T cells (DETCs), has a higher abundance of dermal &#947;&#948; T cells, and varies in immune cell subset localization in comparison to human tissue4. Therefore, experimental findings regarding human skin biology and inflammation benefit from validation with human tissue. While in vitro and organoid culture systems are widely utilized tools to study human tissue, these systems are limited by absent or incomplete immune reconstitution and a lack of connection to peripheral vasculature5. The humanized xenograft skin transplant model aims to allow for therapeutic or biological manipulation of immune and non-immune pathways in human tissues in vivo.
The human skin xenograft model has been utilized to study skin physiology and pharmacology, analyze immune rejection and responses, dissect human skin cancer mechanisms, and understand skin diseases and wound healing6. While applicable to multiple fields of skin research, the xenograft model has lower throughput than in vitro studies and lacks the ease of genetic manipulation employed in mouse models. Time points within this model may range from weeks to months, and successful grafting requires appropriate facilities and equipment to perform these surgeries. However, the xenograft model supplies biological and physiological context to experiments, while organoid culture systems, such as tissue explants, often require replicating a myriad of moving parts, such as exogenous signals, at specific time intervals7. Therefore, this model is best utilized to further validate findings observed in vitro and within mouse models, or for work that is not otherwise biologically feasible. Appropriate use of the xenograft model provides a unique opportunity to study and manipulate intact human tissue in vivo.
Optimization of the xenograft skin transplant model has relied on decades of research to preserve graft integrity over time. Critical to this process is utilizing the non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mouse, which lacks B and T adaptive immune cells, functional NK cells, and has deficiencies in macrophage and dendritic cells8. The immunodeficient nature of these NSG hosts allows for the transplant of human hematopoietic cells, patient-derived cancers, and skin8,9,10. Despite this immunosuppressive host environment, additional suppression of mouse neutrophilic immune responses by anti-GR1 administration is necessary for graft success10. The major roadblocks in transplanting intact tissue are infection, rejection, and difficulty in re-establishing blood flow to the graft, sometimes leading to loss of dermal and epidermal integrity11. Techniques including administration of anti-FR1 and use of appropriate graft depth improve graft survival10. Meticulous optimization makes it possible to perform human xenograft skin transplants on NSG mice with high efficiency and survival rates, ranging from 90%-100%.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher</td>
			<td>SF100-20</td>
			<td>Fixative for histology</td>
		</tr>
		<tr>
			<td>3M Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>surgical glue</td>
		</tr>
		<tr>
			<td>Alexa 700 CD45 monoclonal antibody (Clone 30F11)</td>
			<td>Thermo Fischer</td>
			<td>56-0451-82</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Anti-GR1 clone RB6-8C5</td>
			<td>BioXcell</td>
			<td>BE0075</td>
			<td>Anti-rejection</td>
		</tr>
		<tr>
			<td>APC mouse anti-human CD25&#160; (Clone 2A3)</td>
			<td>BD Biosciences</td>
			<td>340939</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>APC-eFluor 780 anti-human HLA-DR (Clone LN3)</td>
			<td>eBioscience</td>
			<td>47-9956-42</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Autoclave pouches</td>
			<td>VWR&#160;</td>
			<td>89140-800</td>
			<td>For autoclaving tools and paper towels</td>
		</tr>
		<tr>
			<td>Brilliant Violet 60 anti-human CD4 antibody (Clone OKT4</td>
			<td>Biolegend</td>
			<td>317438</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 65 anti-human CD8a antibody (Clone RPA-T8)</td>
			<td>Biolegend</td>
			<td>301042</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Brilliant Violet 711 anti-human CD3 antibody (Clone OKT3)</td>
			<td>Biolegend</td>
			<td>317328</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Buprenex 0.3 mg/mL</td>
			<td>Covetrus</td>
			<td>059122</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Carprofen 50 mg/mL</td>
			<td>Zoetis</td>
			<td>NADA # 141-199</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>4188</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>D42 Dermatome blade</td>
			<td>Humeca</td>
			<td>5.D42BL10</td>
			<td>dermatome (1 blade per sample)</td>
		</tr>
		<tr>
			<td>Dermatome D42</td>
			<td>Humeca</td>
			<td>4.D42</td>
			<td>dermatome</td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Bard-Parker</td>
			<td>371610</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Dissecting T-Pins; 1-1/2 inch, 1000/CS 1.5</td>
			<td>Cole-Parmer</td>
			<td>UX-10915-03</td>
			<td>To pin skin specimen for dermatome</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>medicon</td>
			<td>02.04.10</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-1G</td>
			<td>Skin digestion</td>
		</tr>
		<tr>
			<td>eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization Concentrate and Diluent</td>
			<td>eBioscience</td>
			<td>00-5521-00</td>
			<td>Flow cytometry analysis: Cell Fixation and Permeabilization</td>
		</tr>
		<tr>
			<td>eFluor-450 FOXP3 monoclonal antibody (Clone PCH101)</td>
			<td>eBioscience</td>
			<td>48-4776-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td>Kent</td>
			<td>CL8787-KIT</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Eosin</td>
			<td>Fisher Scientific</td>
			<td>6765040</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>Epredia&#160;Shandon Instant Hematoxylin</td>
			<td>Fisher Scientific</td>
			<td>6765015</td>
			<td>H&#38;E</td>
		</tr>
		<tr>
			<td>FITC anti-human CD45 (Clone HI30)</td>
			<td>Tonbo Biosciences</td>
			<td>35-0459-T100</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>Forceps&#160;</td>
			<td>medicon</td>
			<td>07.60.07</td>
			<td>sample preparation and mouse dissection</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td>Sample preparation</td>
		</tr>
		<tr>
			<td>Heating lamp</td>
			<td>Morganville Scientific</td>
			<td>HL0100</td>
			<td>Post-surgical care</td>
		</tr>
		<tr>
			<td>Heating pads 4&#34; x 10&#34;</td>
			<td>Pristech</td>
			<td>20415</td>
			<td>Surgical heat supply</td>
		</tr>
		<tr>
			<td>Insulin 1cc 12.7 mm syringes</td>
			<td>BD</td>
			<td>329410</td>
			<td>drug administration</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>United States Pharmacopeia (USP)&#160;</td>
			<td>NDC 66794-013-25</td>
			<td>Anesthesia&#160;</td>
		</tr>
		<tr>
			<td>Isoflurane machine</td>
			<td>VetEquip</td>
			<td>911103</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Nair for Men</td>
			<td>Nair</td>
			<td>&#8206; 10022600588556</td>
			<td>hair removal</td>
		</tr>
		<tr>
			<td>Neomycin and Polymyxin Bisulfates and Bacitracin Zinc Ophthalmic ointment</td>
			<td>Dechra</td>
			<td>&#160;NDC 17478-235-35</td>
			<td>eye ointment to prevent drying</td>
		</tr>
		<tr>
			<td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>Mice</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>Kleenex</td>
			<td>100848</td>
			<td>May be autoclaved for sterile surfaces</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td>Semitransparent sealing film</td>
		</tr>
		<tr>
			<td>PE mouse anti-human CD127 (Clone HIL-7R-M21)</td>
			<td>BD Biosciences</td>
			<td>557938</td>
			<td>Flow cytometry analysis: Surface protein staining</td>
		</tr>
		<tr>
			<td>PE-Cy-7 mouse anti-Ki-67 (Clone B56)</td>
			<td>BD Biosciences</td>
			<td>561283</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>PerCP-eFluor-710 CD152 (CTLA-4) monoclonal antibody (Clone 14D3)</td>
			<td>eBioscience</td>
			<td>46-1529-42</td>
			<td>Flow cytometry analysis: Intracellular protein staining</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer 10x</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td>Flow cytometry analysis: Intracellular protein staining buffer</td>
		</tr>
		<tr>
			<td>Petri Dish 150 mm</td>
			<td>Corning</td>
			<td>430597</td>
			<td>Sample storage</td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>Fisherbrand</td>
			<td>22-305-654</td>
			<td>Site preparation</td>
		</tr>
		<tr>
			<td>Providone-Iodine Swab stick</td>
			<td>PDI</td>
			<td>S41350</td>
			<td>Site sterilization</td>
		</tr>
		<tr>
			<td>Soft-Feed and Oral Hydration (Napa Nectar)</td>
			<td>Se Lab Group Inc</td>
			<td>NC9066511&#160;</td>
			<td>For supplementing poorly recovering mice post-surgery</td>
		</tr>
		<tr>
			<td>Specimen Collection Cups</td>
			<td>Fisher Scientific</td>
			<td>22-150-266</td>
			<td>sample storage</td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td>skin preparation</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Media for sample storage</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-02</td>
			<td>For drug dilution</td>
		</tr>
		<tr>
			<td>Tegaderm Film 4&#8221; x 43/4&#8221;&#160;</td>
			<td>3M</td>
			<td>1626</td>
			<td>transparent film wound dressing</td>
		</tr>
		<tr>
			<td>Vaseline Petrolatum Gauze 3&#8221; x 8&#8221;&#160;</td>
			<td>Kendall</td>
			<td>414600</td>
			<td>wound dressing</td>
		</tr>
		<tr>
			<td>Violet 510 Ghost Dye&#160;</td>
			<td>Tonbo Biosciences</td>
			<td>13-0870-T100</td>
			<td>Flow cytometry analysis: Viability dye</td>
		</tr>
	</tbody>
</table>

xenograft skin model to manipulate human immune responses in vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Human skin xenografts were performed on NSG mice inside a super-barrier animal facility. Success was defined by the prolonged graft and mouse survival and behavioral health of mice post-transplant. Poor survival during the week following surgery was initially observed as the biggest barrier to experimental success, with up to 50% of mice requiring euthanasia. Improving sterile technique and better support of mouse body temperatures during and immediately after surgery increased surgical survival consistently to over 80% ...

Watch this Scientific Journal Video about Xenograft Skin Model to Manipulate Human Immune Responses In Vivo at JoVE.com

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

xenograft-skin-model-to-manipulate-human-immune-responses-in-vivo

Research

JoVE Journal

Immunology and Infection

2.7K Views.  UCSF. The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report. 

Video: Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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

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

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

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

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.