This work was funded by the American Lung Association (K.J.W.), the Memorial Eugene Kenney fund awarded to T.A.W. and K.J.W., generous start-up support from the University of Utah for K.J.W., and a Department of Veterans Affairs award to T.A.W. (VA I01BX0003635). T.A.W. is the recipient of a Research Career Scientist Award (IK6 BX003781) from the Department of Veterans Affairs. The authors wish to acknowledge editorial assistance from Ms. Lisa Chudomelka. The authors thank the UNMC Flow Cytometry core for their support in collecting the flow cytometry data generated for this manuscript.

All methods described here were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Nebraska Medical Center (UNMC) and the University of Utah.
1. Setup and Preparation of Reagents
<ol>
	<li>Prepare complete RPMI (Roswell Park Memorial Institute) media.
	<ol>
		<li>Add 10 mL of heat-inactivated fetal bovine serum (FBS) to 90 mL of RPMI.</li>
		<li>Add 1 mL of 100x Penicillin-Streptomycin-Glutamine to 100 mL of 10% FBS RPMI.</li>
	</ol>
	</li>
	<li>Prepare ILC2 Expansion Media.
	<ol>
		<li>Add IL-2 and IL-33 (20 ng/mL each cytokine) to 10 mL of complete RPMI.</li>
		<li>If stock cytokines are 10 &#181;g/mL, pipette 20 &#181;L of IL-2 and IL-33 into a 15 mL tube containing 10 mL of complete RPMI media.</li>
	</ol>
	</li>
	<li>Prepare Lung Dissociation Medium.
	<ol>
		<li>Add 50 mg of type 1 collagenase to 250 mL of unsupplemented RPMI.</li>
		<li>Add 2.5 mL of 100x penicillin-streptomycin-glutamine to the 250 mL of media in step 1.3.1.</li>
		<li>Gently mix the media to ensure the type 1 Collagenase is completely dissolved before use.</li>
	</ol>
	</li>
	<li>Prepare Serum-free RPMI.
	<ol>
		<li>Dilute 1 g of lyophilized bovine serum albumin (BSA) in 200 mL of RPMI.</li>
		<li>Add 2 mL of 100x penicillin-streptomycin-glutamine.</li>
		<li>Gently mix the media to ensure the BSA is completely dissolved in the media before use.</li>
	</ol>
	</li>
	<li>Prepare Migration Medium with CCL17.
	<ol>
		<li>Acquire 10 mL of serum-free RPMI and add CCL17 [50 ng/mL].
		<ol>
			<li>If stock CCL17 is 10 &#181;g/mL, add 50 &#181;L of CCL17 stock to 10 mL of serum-free RPMI media.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare Migration Medium with CCL22.
	<ol>
		<li>Acquire 10 mL of serum-free RPMI and add CCL22 [50 ng/mL].</li>
		<li>If stock CCL22 is 10 &#181;g/mL, add 50 &#181;L of CCL22 stock to 10 mL of serum-free RPMI media.</li>
	</ol>
	</li>
	<li>Prepare CCR4 Antibody Staining Cocktail.
	<ol>
		<li>For 10 tests total, add 5 &#181;L of each of the following antibodies to a 5 mL tube: anti-mouse CCR4, CD19, CD11b, CD45, ST2, and ICOS. 
		NOTE: Example of how to make antibody cocktail for 10 samples: 0.5 &#181;L of each antibody x 10 samples = 5 &#181;L of each of the antibodies listed in 1.7.1.</li>
		<li>For 10 tests total, add 2.5 &#181;L of the following antibodies to the antibody cocktail from step 1.7.1: anti-mouse CD3, CD11c, and NK1.1. 
		NOTE: Example to complete the antibody cocktail for 10 samples: 0.25 &#181;L x 10 samples = 2.5 &#181;L of CD3, CD11c and NK1.1 antibodies.</li>
		<li>Store the CCR4 Antibody Staining Cocktail at 4 &#176;C until ready to add to the samples. Discard antibody cocktail after 1 week if not used.</li>
	</ol>
	</li>
	<li>Prepare 1x Stabilizing Fixative.
	<ol>
		<li>Add 10 mL of deionized-distilled water to 5 mL of 3x stabilizing fixative concentrate</li>
	</ol>
	</li>
</ol>
2. Preparation of Allergen-challenged BALB/c Mice
NOTE: Male and female BALB/c mice were purchased from Charles River (UNMC) or Jackson Laboratories (University of Utah) at 6 to 8 weeks of age.
<ol>
	<li>After acclimation (1 week), sensitize all animals to OVA.
	<ol>
		<li>Combine 100 &#181;g/mL OVA adsorbed to aluminum hydroxide (20 mg/mL) in a 5 mL polystyrene tube.</li>
		<li>Mix the tube and immediately draw 500 &#181;L of the OVA-alum suspension into a 1 mL, 28 G syringe (insulin syringe).</li>
		<li>Place a mouse in a bell jar containing isoflurane (1&#8211;2 mL under a false floor, so that the animal is not standing directly in isoflurane). Allow the mouse to go under anesthesia for approximately 1&#8211;2 min, or until the rate of respiration drops.</li>
		<li>Quickly pick up the mouse by the fur on the back and shoulders and inject 100 &#181;L of OVA-alum intraperitoneally per mouse21,22.</li>
		<li>Place the mouse back in its cage and watch to make sure they regain mobility; this should occur within 2&#8211;5 min.</li>
	</ol>
	</li>
	<li>Seven days after sensitization, subject all animals to intranasal (i.n.) 1.5% OVA diluted in sterile saline in a nebulization chamber (Data Sciences International) for 20 min.
	<ol>
		<li>Remove mice from their cages and place them in the animal nebulization chamber. Close the lid on the chamber.</li>
		<li>Attach the nebulization hose to the input spout on the nebulization chamber.</li>
		<li>Add 30 mL of 1.5% OVA diluted in sterile saline to the nebulization cup on the nebulizer.</li>
		<li>Turn on the nebulizer and allow the chamber to fill with mist for 20 min.</li>
		<li>Turn off the nebulizer and let the mist settle.</li>
		<li>Return animals to their cages.</li>
	</ol>
	</li>
	<li>Repeat step 2.2 for a total of 5 times, on 5 consecutive days, to induce allergic inflammation.</li>
</ol>
3. Isolation of CD4+ T Cells from Spleens and Lungs of OVA-challenged Mice
<ol>
	<li>Humanely euthanize all OVA-treated male and female animals by CO2 asphyxiation according to approved IACUC protocols, using 2 to 3 animals per group, per experiment.</li>
	<li>Excise lungs and spleens from animals and place tissues in separate dissociation tubes based on the tissue type and sex of the animal23.</li>
	<li>Dissociate lung tissue in 500 &#181;L of Lung Dissociation media (25 U/mL; collagenase, type 1) in the automated tissue dissociator using the &#8216;lung&#8217; protocol.
	<ol>
		<li>Repeat 3.2 and 3.3 a total of two times.</li>
	</ol>
	</li>
	<li>Dissociate spleen tissue in 500 &#181;L of complete RPMI media using the &#8216;spleen&#8217; protocol on the automated tissue dissociator. 
	NOTE: The remaining steps should be performed in a Biological Safety cabinet using sterile technique.</li>
	<li>Rinse the dissociation tubes containing lung and spleen homogenates with 5 mL of additional Lung Dissociation media or Complete RPMI, respectively.</li>
	<li>Filter cell suspensions through a 40 &#181;m cell strainer and collect into 50 mL conical tubes.</li>
	<li>Incubate lung homogenates for 15&#8211;30 min in a 37 &#176;C incubator with 5% CO2 to further dissociate lung tissue.</li>
	<li>Add 5 mL of Complete RPMI to the lung and spleen homogenates and pellet the cells at the bottom of the 50 mL tubes using centrifugation; 378 x g at room temperature (RT) for 5 min.</li>
	<li>Combine splenocytes and lung cells into a single 50 mL conical tube and determine total cell counts using the automated cell counter.</li>
	<li>Adjust male and female cell suspensions to 1 x&#160;108 cells/mL in separation buffer and add to a 5 mL polystyrene tube. 
	NOTE: The Enrichment Protocol can be adjusted up to 14 mL polystyrene tubes when more cells are acquired from spleen and lungs. This protocol was designed for tissues from 2&#8211;3 mice per group; therefore a 5 mL tube should suffice.</li>
	<li>Use approximately two thirds of the total cells for ILC2 isolation according to the ILC2 enrichment protocol.
	<ol>
		<li>Add antibody cocktail (50 &#181;L/mL) to the cell suspension and incubate for 5 min&#160;at RT.</li>
		<li>Vortex rapid spheres for 30 s and add to the sample at a rate of 75 &#181;L/mL of cell suspension. Gently mix and incubate for 5 min at RT.</li>
		<li>Top the tube up to 3 mL total volume with separation buffer and place in the 8-chamber easy separation magnet. Incubate for 3 min&#160;at RT.</li>
		<li>Tip the magnet forward (away from the sphere-antibody-cell complexes adhered to the back of the tube) and pipette off the cell suspension into a clean 5 mL tube.</li>
		<li>Add 1.5 mL of complete RPMI to the tubes and centrifuge at 378 x g for 5 min at RT.</li>
		<li>Pour off the media from the cell pellet and resuspend the ILC2 at 1 x 107 cells per mL.</li>
		<li>Place 100 uL of male and female ILC2 per well in a U-bottom, 96-well plate and add 100 &#181;L of ILC2 Expansion Media to each well.</li>
		<li>Incubate the cells for 4&#8211;5 days to expand the ILC2.</li>
		<li>Collect the ILC2 into a 5 mL tube and add up to 4.5 mL of serum-free RPMI. Centrifuge the cells at 378 x g for 5 min at RT.</li>
		<li>Count cells using a hemacytometer and resuspend at 1 x 107 ILC2 per mL in serum-free RPMI.</li>
	</ol>
	</li>
	<li>Use the remaining cells for the CD4+ T cell isolation procedure, which is conducted according to the mouse CD4+ T cell isolation protocol with few modifications.
	<ol>
		<li>Add rat serum (50 &#181;L/mL) to the CD4 T cell enrichment suspension.</li>
		<li>Add Isolation cocktail (50 &#181;L/mL) to the sample and incubate for 10 min at RT.</li>
		<li>Vortex rapid spheres for 30 s and add to the sample at a rate of 75 &#181;L/mL.</li>
		<li>Gently mix the cell suspension and incubate for 2.5 min and RT</li>
		<li>Top the samples up to 3 mL and place the 5 mL tubes into the 8-chamber easy separation magnet and incubate for 5 min at RT.</li>
		<li>Tip the magnet forward and pipette the cell suspension into a clean 5 mL tube.</li>
		<li>Add 1.5 mL of serum-free RPMI to the tubes and centrifuge at 378 x g for 5 min at RT.</li>
		<li>Pour off the media from the cell pellet and resuspend the CD4+ T cells at 1 x 107 cells per mL in serum-free RPMI.</li>
	</ol>
	</li>
</ol>
4. Determine CCR4 Expression on CD4+ T Cells and Group 2 Innate Lymphoid Cells (ILC2) from OVA-challenged Animals by Flow Cytometry
NOTE: The following steps may be performed on an open bench top as they are non-sterile techniques.
<ol>
	<li>Acquire approximately 1&#8211;2.5 x 105 ILC2 cells from step 3.11.10 and 1&#8211;2.5 x 106 CD4+ T cells from step 3.12.8 in separate 5 mL tubes.
	<ol>
		<li>Keep an additional tube of at least 5.0 x 104 CD4+ T cells as an unstained control.</li>
	</ol>
	</li>
	<li>Suspend the cells in 100&#8211;200 &#181;L of FACS buffer and add 1 &#181;L of Fc Block to every tube, then incubate on ice (or in a 4 &#176;C refrigerator) for 10 min.</li>
	<li>Add 1&#8211;2 mL of FACS Buffer to every tube and centrifuge at 378 x g for 5 min at RT.</li>
	<li>Pour off the supernatant and resuspend the cells in 100&#8211;200 &#181;L of FACS Buffer.</li>
	<li>Add 37.5 &#181;L of the CCR4 Antibody Staining Cocktail to the cell suspension in every tube except the tube containing the &#8216;unstained&#8217; cells.</li>
	<li>Incubate the tubes on ice, or in a 4 &#176;C refrigerator, for 20&#8211;30 min.</li>
	<li>Add 1&#8211;2 mL of FACS Buffer to every tube and centrifuge at 378 x g for 5 min at RT.</li>
	<li>Pour off the supernatant.</li>
	<li>Repeat steps 4.7 and 4.8.</li>
	<li>Add 250&#8211;300 &#181;L of 1x stabilizing fixative (see Table of Materials) to every tube.</li>
	<li>Prepare single-colored bead controls for each antibody in the CCR4 antibody cocktail according to the protocol provided with the compensation beads.
	<ol>
		<li>Vortex the compensation beads.</li>
		<li>Add one drop of the beads to each single-colored control tube.</li>
		<li>Add 1 &#181;L of each antibody in the CCR4 Antibody cocktail to its own labeled tube.</li>
		<li>Gently mix and incubate for 10 min in a 4 &#176;C refrigerator or on ice.</li>
		<li>Add 1&#8211;2 mL of FACS Buffer to all the tubes and centrifuge at 378 x g for 5 min at RT.</li>
		<li>Pour off the supernatant and resuspend the beads in 200 &#181;L of FACS buffer.</li>
		<li>Refrigerate the single-colored controls until all the samples are stained and ready to be analyzed on the flow cytometer.</li>
	</ol>
	</li>
	<li>Analyze the unstained cells, single-colored controls and the experimental samples on the flow cytometer within 24 h of fixation.</li>
</ol>
5. Ex Vivo Transmigration Procedure
NOTE: The following steps should be performed in a Biological Safety Cabinet, as they require sterile technique.
<ol>
	<li>Acquire the ILC2 from step 3.11.10 and CD4 T cells from step 3.12.8 and determine the number of transwell inserts needed for the experiment. 
	NOTE: Example: For 1.2 mL of enriched CD4 T cells from step 3.12.8, multiply 1.2 x 1,000 &#181;L = 1,200 &#181;L; then divide 1,200 uL by 100 &#181;L = 12, the number of inserts needed for CD4 T transmigration.</li>
	<li>Gently move the 3 &#181;m transwell inserts from the middle rows of a 24-well plate.</li>
	<li>Add 500 &#181;L of migration media with CCL17 to approximately one-third of the wells.</li>
	<li>Add 500 &#181;L of migration media with CCL22 to another one-third of the wells.</li>
	<li>Add 500 &#181;L of serum-free RPMI containing no chemokine to the last one-third of the wells.</li>
	<li>Clearly label the lid on the plate with the appropriate transmigration media placed in the bottom wells.</li>
	<li>Place the transwell inserts back into the wells containing the various treatments.</li>
	<li>Gently add 100 &#181;L of CD4 T cells or ILC2 to the top well of each insert. Do not mix or pipette the cell suspension up and down in the transwells, as this may confound the results of the experiment.</li>
	<li>Clearly label the lid of the plate with the cell type placed in each well and write the date and time of day that the cells were added.</li>
	<li>Repeat steps 5.2 to 5.9 until all the cells have been placed in transwell inserts with media.</li>
	<li>Gently place the plate in a 37 &#176;C incubator with 5% CO2 for 48 h. Minimize contact with the plate over the incubation period.</li>
</ol>
6. Quantification of Ex Vivo Transmigration
<ol>
	<li>Gently remove the plate from the incubator and remove all the transwell inserts from the middle rows into the empty wells just above or below.</li>
	<li>Collect the cells from the bottom and top wells of the transwell inserts into tubes labeled with TOP or BOTTOM, with CCL17, CCL22, or media, with the cell type, and with the replicate number (at least 3 replicates per experiment).</li>
	<li>Rinse the bottom wells with 500 &#181;L of 1x PBS and collect this rinse into the corresponding tube.</li>
	<li>Rinse the top wells with 250 &#181;L of 1x PBS and collect this rinse into the corresponding tube.</li>
	<li>Pellet the cells by centrifugation at 378 x g for 5 min at RT.</li>
	<li>Gently pipette off all supernatant from the cell pellet.</li>
	<li>Resuspend T cells and ILC2 into 50 &#181;L of 1x PBS.</li>
	<li>Take 10 &#181;L of the cell suspensions and add to 90 &#181;L of 0.4% trypan blue.</li>
	<li>Count the cells on the automated cell counter.
	<ol>
		<li>Record %viability.</li>
		<li>Record the cell count per mL for each sample.</li>
		<li>Determine the total number of cells per treatment in the top and the bottom chamber; record the cell counts.</li>
	</ol>
	</li>
</ol>

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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=alphabeta+T+cells+and+a+mixed+Th1/Th17+response+are+important+in+organic+dust-induced+airway+disease.">alphabeta T cells and a mixed Th1/Th17 response are important in organic dust-induced airway disease.</a> Annals of Allergy, Asthma, Immunology: Official Publication of the American College of Allergy, Asthma, Immunology. 109 (4), 266-273 (2012).</li><li>Matsuo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CCR4+antagonist+ameliorates+atopic+dermatitis-like+skin+lesions+induced+by+dibutyl+phthalate+and+a+hydrogel+patch+containing+ovalbumin.">A CCR4 antagonist ameliorates atopic dermatitis-like skin lesions induced by dibutyl phthalate and a hydrogel patch containing ovalbumin.</a> Biomedical Pharmacotherapy. 109, 1437-1444 (2019).</li><li>Mikhak, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+CCR4+and+CCR8+to+antigen-specific+T(H)2+cell+trafficking+in+allergic+pulmonary+inflammation.">Contribution of CCR4 and CCR8 to antigen-specific T(H)2 cell trafficking in allergic pulmonary inflammation.</a> The Journal of Allergy and Clinical Immunology. 123 (1), 67-73 (2009).</li><li>Monticelli, L. 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=Innate+lymphoid+cells+promote+lung-tissue+homeostasis+after+infection+with+influenza+virus.">Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus.</a> Nature Immunology. 12 (11), 1045-1054 (2011).</li><li>Saenz, S. 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=IL25+elicits+a+multipotent+progenitor+cell+population+that+promotes+T(H)2+cytokine+responses.">IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses.</a> Nature. 464 (7293), 1362-1366 (2010).</li><li>Hoyler, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+GATA-3+controls+cell+fate+and+maintenance+of+type+2+innate+lymphoid+cells.">The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells.</a> Immunity. 37 (4), 634-648 (2012).</li><li>Nakajima, H., Shores, E. W., Noguchi, M., Leonard, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+common+cytokine+receptor+gamma+chain+plays+an+essential+role+in+regulating+lymphoid+homeostasis.">The common cytokine receptor gamma chain plays an essential role in regulating lymphoid homeostasis.</a> The Journal of Experimental Medicine. 185 (2), 189-195 (1997).</li><li>Warren, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RSV-specific+anti-viral+immunity+is+disrupted+by+chronic+ethanol+consumption.">RSV-specific anti-viral immunity is disrupted by chronic ethanol consumption.</a> Alcohol. , (2016).</li><li>Warren, K. J., Poole, J. A., Sweeter, J. M., DeVasure, J. M., Wyatt, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+association+between+MMP-9+and+impaired+T+cell+migration+in+ethanol-fed+BALB/c+mice+infected+with+Respiratory+Syncytial+Virus-2A.">An association between MMP-9 and impaired T cell migration in ethanol-fed BALB/c mice infected with Respiratory Syncytial Virus-2A.</a> Alcohol. , (2018).</li><li>Molteni, 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=A+novel+device+to+concurrently+assess+leukocyte+extravasation+and+interstitial+migration+within+a+defined+3D+environment.">A novel device to concurrently assess leukocyte extravasation and interstitial migration within a defined 3D environment.</a> Lab on a Chip. 15 (1), 195-207 (2015).</li><li>Bersini, 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=Human+in+vitro+3D+co-culture+model+to+engineer+vascularized+bone-mimicking+tissues+combining+computational+tools+and+statistical+experimental+approach.">Human in vitro 3D co-culture model to engineer vascularized bone-mimicking tissues combining computational tools and statistical experimental approach.</a> Biomaterials. 76, 157-172 (2016).</li><li>Jeon, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+3D+vascularized+organotypic+microfluidic+assays+to+study+breast+cancer+cell+extravasation.">Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (1), 214-219 (2015).</li></ol>

The authors have no financial disclosures or conflicts of interest to disclose.

Herein, we present a well-established method for assessing chemokine-induced migration of lymphocytes in an ex vivo transmigration system. There are several critical steps in the protocol, the first of which is verifying the expression of the correct chemokine receptor on the immune cells in the experiment. In our hands, we chose CCR4 because of the body of literature that highlights the importance of CCR4 on Th2 helper T cells in allergic inflammation. Ovalbumin-induced inflammation was shown previously to be limited by...

This original transmigration method was presented by Stephen Boyden in 1962 in the Journal of Experimental Medicine1. Much of what we know about chemotaxis and chemokinetics would not be possible without the development of the Boyden chamber. Prior to the discovery of the first chemokine in 1977, ex vivo transmigration systems were used to learn about serum-factors that could arrest cellular movement in macrophages while amplifying cellular motility in neutrophils1,2. A massive wealth of knowledge has been developed regarding immune cell migration, and to date, 47 chemokines have now been discovered with 19 corresponding receptors3,4. In addition, multitudes of inhibitors/enhancers of these chemokine pathways have undergone development for therapeutic purposes5,6,7,8. Many of those compounds have been tested in similar transmigration chambers to understand direct interactions between the compounds and immune cell responsiveness to a given chemokine9.
Transmigration, or diapedesis, into inflamed tissue is an essential process to a healthy inflammatory response to clear infection10,11. A Boyden chamber, transmigration system, or transwell apparatus are generally composed of two chambers separated by a porous membrane1,12. The bottom chamber most often holds media containing the chemokine of interest, while leukocytes are placed in the top chamber. The size of the pore in the membrane can be selected based on the size of the cell of interest. For this project, we selected a 3 &#181;m porous membrane, as lymphoid cells are 7-20 &#181;m in size, depending on the stage of cellular development. This pore size ensures that these cells are not passively falling through the pores, but that they are actively migrating in response to the chemokine gradient.
The major advantage of this protocol is its cost effectiveness. In vivo transmigration is difficult because it requires extensive training in animal handling and surgery, and often involves high-powered microscopy that is not always available to a researcher. Cost effective screening of compounds thought to enhance or inhibit transmigration can be accomplished in advance of in vivo imaging. Because the transmigration system is tightly controlled, cells may be treated initially then added to the transwell apparatus, or, vice versa, the chemokine may be treated first with a chemokine inhibitor then cells added to the transwell apparatus. Lastly, endothelial cells and/or basement membrane proteins can be added to the bottom of the transwell insert 1-2 days prior to the transmigration experiment to understand the involvement of these barrier cells in chemokinetics. Again, these manipulations of the system provide a powerful means of determining important information about the effectiveness of a given compound in advance of more complicated in vivo studies.
Utilizing a transmigration chamber system is an effective way to assess lymphocyte mobility under various in vivo and in vitro conditions12,13,14. Herein, we describe an optimized method for assessing ex vivo lymphocyte responsiveness to chemokines in a transmigration chamber. In this example experiment, CD4+ T cells and group 2 innate lymphoid cells (ILC2) were isolated from male and female, BALB/c mice following OVA-allergen exposure. A hypothesis was generated that CCR4+ CD45+ Lineage- (LIN-) ILC2 from allergen-challenged mice would migrate more efficiently towards CCL17 and CCL22 than CCR4+ CD4+ T helper cells. CCL17 and CCL22 are chemokines commonly produced by dendritic cells and macrophages of the M2 (allergic) phenotype, among other cells, in allergy15,16. CCL17 and CCL22 can be thought of as biomarkers of allergic inflammation as they are readily detected in the lungs during airway exacerbations16,17,18. Importantly, CCR4 expression is elevated in comparison to untreated controls, as revealed in bioinformatic data generated from ILC2 isolated from house dust mite treated animals, and similarly ILC2 from na&#239;ve animals treated ex vivo with IL-33 (allergen-promoting innate cytokine) upregulates CCR419,20. Furthermore, according to data for ILC2 in the Immunological Genome Project database (www.immgen.org), CCR4 mRNA is highly expressed in these innate immune cells. To date, little is known regarding trafficking of ILC2 into tissues, but it is likely that the ILC2 and CD4+ T cells use similar chemokines and receptors for chemotaxis and chemokinetics as they express similar transcription factors and receptors. Thus, we compared CCL17 versus CCL22 responsiveness, of ILC2 and CD4+ T lymphocytes, from both male and female, OVA-challenged animals.

<table>
	<tbody>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>15250061</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD Bioscience</td>
			<td>329424</td>
			<td>U-100 Syringes Micro-Fine 28G 1/2&#34; 1cc</td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin, L-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td>Dilute to 1x in RPMI media</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td>polypropylene tubes</td>
		</tr>
		<tr>
			<td>3 um transwell inserts</td>
			<td>Genesee Scientific</td>
			<td>25-288</td>
			<td>24-well plate containing 12 transwell inserts</td>
		</tr>
		<tr>
			<td>3x stabilizing fixative</td>
			<td>BD Pharmigen</td>
			<td>338036</td>
			<td>Prepare 1x solution according to manufacturers protocol</td>
		</tr>
		<tr>
			<td>5 mL polystyrene tubes</td>
			<td>STEM Cell Technologies</td>
			<td>38007</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-106</td>
			<td>polypropylene tubes</td>
		</tr>
		<tr>
			<td>8-chamber easy separation magnet</td>
			<td>STEM Cell Technologies</td>
			<td>18103</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Life Technologies Corporation</td>
			<td>A1049201</td>
		</tr>
		<tr>
			<td>Advanced cell strainer, 40 um</td>
			<td>Genesee Scientific</td>
			<td>25-375</td>
			<td>nylon mesh, 40 micron strainers</td>
		</tr>
		<tr>
			<td>Aluminum Hydroxide, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>239186-25G</td>
			<td>20 mg/mL</td>
		</tr>
		<tr>
			<td>anti- mouse CCR4; APC-conjugated</td>
			<td>Biolegend</td>
			<td>131211</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>BD Pharmigen</td>
			<td>557396</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD11c; PE eFluor 610</td>
			<td>Thermo-Fischer Scientific</td>
			<td>61-0114-82</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32, Fc block</td>
			<td>BD Pharmigen</td>
			<td>553141</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD19; APC-eFluor 780 conjugated</td>
			<td>Thermo-Fischer Scientific</td>
			<td>47-0193-82</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD3; PE Cy 7-conjugated</td>
			<td>BD Pharmigen</td>
			<td>552774</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD45; PE conjugated</td>
			<td>BD Pharmigen</td>
			<td>56087</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse ICOS (CD278)</td>
			<td>BD Pharmigen</td>
			<td>564070</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse NK1.1 (CD161); FITC-conjugated</td>
			<td>BD Pharmigen</td>
			<td>553164</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugated</td>
			<td>Biolegend</td>
			<td>145311</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>BIORAD</td>
			<td>1450102</td>
		</tr>
		<tr>
			<td>Automated Dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Lyophilized Powder</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-10G</td>
			<td>0.5% in serum-free RPMI</td>
		</tr>
		<tr>
			<td>Cell Counter Clides</td>
			<td>BIORAD</td>
			<td>1450015</td>
		</tr>
		<tr>
			<td>Chicken Egg Ovalbumin, Grade V</td>
			<td>Sigma-Aldrich</td>
			<td>A5503-10G</td>
			<td>500 ug/mL</td>
		</tr>
		<tr>
			<td>Collagenase, Type 1, Filtered</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLSS-1, purchase as 5 X 50 mg vials (LS004216)</td>
			<td>25 U/mL in RPMI</td>
		</tr>
		<tr>
			<td>compensation beads</td>
			<td>Affymetrix</td>
			<td>01-1111-41</td>
			<td>1 drop per contol tube</td>
		</tr>
		<tr>
			<td>Dissociation Tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-096-335</td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td>BD Pharmigen</td>
			<td>554657</td>
			<td>1x PBS + 2% FBS, w/ sodium azide; stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Heat Inactivated-FBS</td>
			<td>Genesee Scientific</td>
			<td>25-525H</td>
			<td>10% in complete RPMI &#38; ILC2 Expansion Media</td>
		</tr>
		<tr>
			<td>mouse CCL17</td>
			<td>GenScript</td>
			<td>Z02954-20</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>mouse CCL22</td>
			<td>GenScript</td>
			<td>Z02856-20</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>mouse CD4+ T cell enrichment kit</td>
			<td>STEM Cell Technologies</td>
			<td>19852</td>
		</tr>
		<tr>
			<td>mouse IL-2</td>
			<td>GenScript</td>
			<td>Z02764-20</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>mouse ILC2 enrichment kit</td>
			<td>STEM Cell Technologies</td>
			<td>19842</td>
		</tr>
		<tr>
			<td>mouse recombinant IL-33</td>
			<td>STEM Cell Technologies</td>
			<td>78044</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Life Technologies Corporation</td>
			<td>22400071</td>
		</tr>
		<tr>
			<td>Separation Buffer</td>
			<td>STEM Cell Technologies</td>
			<td>20144</td>
			<td>1 X PBS + 2% FBS; stored at 4C</td>
		</tr>
		<tr>
			<td>small animal nebulizer and chamber</td>
			<td>Data Sciences International</td>
		</tr>
		<tr>
			<td>sterile saline</td>
			<td>Baxter</td>
			<td>2F7124; NDC 0338-0048-04</td>
			<td>0.9% Sodium Chloride</td>
		</tr>
	</tbody>
</table>

assessment of lymphocyte migration in an ex vivo transmigration system

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

CCR4 expression on CD4+ T cells and ILC2.
For the success of the ex vivo transmigration experiment, it is imperative to determine whether the lymphocytes are responsive to CCL17 and CCL22 through CCR4; therefore, we determined CCR4 expression on both CD4+ T cells and ILC2 by flow cytometry. While it is well known that OVA-specific CD4+ helper T cells express CCR4, less is known of the expression of CCR4 on ILC2. Figure 1 show...

Watch this Scientific Journal Video about Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System at JoVE.com

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

assessment-of-lymphocyte-migration-in-an-ex-vivo-transmigration-system

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JoVE Journal

Immunology and Infection

6.7K Views.  University of Utah. In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Video: Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.