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><td></td></tr><tr><td>1 mL syringe</td><td>BD Bioscience</td><td>329424</td><td>U-100 Syringes Micro-Fine 28 G 1/2&quot; 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 &amp;mu;m 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><td></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><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Life Technologies Corporation</td><td>A1049201</td><td></td></tr><tr><td>Advanced cell strainer, 40 &amp;mu;m</td><td>Genesee Scientific</td><td>25-375</td><td>nylon mesh, 40 &amp;mu;m 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 &amp;mu;g/test</td></tr><tr><td>anti-mouse CD11b</td><td>BD Pharmigen</td><td>557396</td><td>0.5 &amp;mu;g/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 &amp;mu;g/test</td></tr><tr><td>anti-mouse CD16/32, Fc block</td><td>BD Pharmigen</td><td>553141</td><td>0.5 &amp;mu;g/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 &amp;mu;g/test</td></tr><tr><td>anti-mouse CD3; PE Cy 7-conjugated</td><td>BD Pharmigen</td><td>552774</td><td>0.25 &amp;mu;g/test</td></tr><tr><td>anti-mouse CD45; PE conjugated</td><td>BD Pharmigen</td><td>56087</td><td>0.5 &amp;mu;g/test</td></tr><tr><td>anti-mouse ICOS (CD278)</td><td>BD Pharmigen</td><td>564070</td><td>0.5 &amp;mu;g/test</td></tr><tr><td>anti-mouse NK1.1 (CD161); FITC-conjugated</td><td>BD Pharmigen</td><td>553164</td><td>0.25 &amp;mu;g/test</td></tr><tr><td>anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugated</td><td>Biolegend</td><td>145311</td><td>0.5 &amp;mu;g/test</td></tr><tr><td>Automated Cell Counter</td><td>BIORAD</td><td>1450102</td><td></td></tr><tr><td>Automated Dissociator</td><td>MACS Miltenyi Biotec</td><td>130-093-235</td><td></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><td></td></tr><tr><td>Chicken Egg Ovalbumin, Grade V</td><td>Sigma-Aldrich</td><td>A5503-10G</td><td>500 &amp;mu;g/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><td></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 &amp;deg;C</td></tr><tr><td>Heat Inactivated-FBS</td><td>Genesee Scientific</td><td>25-525H</td><td>10% in complete RPMI &amp;amp; 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&lt;sup&gt;+&lt;/sup&gt; T cell enrichment kit</td><td>STEM Cell Technologies</td><td>19852</td><td></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><td></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><td></td></tr><tr><td>Separation Buffer</td><td>STEM Cell Technologies</td><td>20144</td><td>1x PBS + 2% FBS; stored at 4 &amp;deg;C</td></tr><tr><td>Small animal nebulizer and chamber</td><td>Data Sciences International</td><td></td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.8K 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 ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Bioengineering

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.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous ...

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Biology

The Transwell Migration Assay

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants.
This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.

The Transwell Migration Assay and Chemo-attractants

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer.  To quantify cell migration, a simple ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Study of Cell Migration in Microfabricated Channels

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

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

The Transwell Migration Assay