This work was supported in part by the National Institutes of Health grants R21 AI113000 (W.S.M) and R01 AI123392 (Y.F.).

All the animals used in the experiments were housed in the animal facility at Allegheny-Singer Research Institute under the protocol reviewed and approved by the Institutional Animal Care and Use Committee of the Allegheny Health Network/Allegheny Singer Research Institute.
1. Digesting the Thymus with Collagenase
<ol>
	<li>Harvest thymus glands.
	<ol>
		<li>Euthanize 3-5 week-old C57BL6/J mice in a carbon dioxide (CO2) chamber to harvest thymus glands. In detail, place the mice in the chamber under CO2 induction for 3-5 min and confirm death by verifying cardiac or respiratory arrest.</li>
		<li>Lay the carcass on its back and wet the body with 70% ethanol. Make a small horizontal incision with a sharp, straight dissecting scissors on its abdomen through the skin. Pull the skin on both ends (head and legs) so that it is turned inside out.</li>
		<li>Make a horizontal cut with the dissecting scissors just under the lowest rib and cut through the diaphragm sideways. Hold the xiphoid process with a forceps and cut the middle of the ribs proceeding up on both sides. Pull up the ribs/sternum so that the thymus is clearly visible. 
		Note: Thymus lies directly on top of the heart, consists of 2 lobes and is of a white translucent color.</li>
		<li>Using a curved forceps, gently scoop and pull the thymus lobes off of the connective tissue and transfer in washing solution (see List of Materials).</li>
	</ol>
	</li>
	<li>Prepare digestion solution by mixing 9 ml RPMI-1640 (Roswell Park Memorial Institute medium-1640), 0.025 mg/ml purified collagenase, 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and 0.2 mg/ml DNase I in a 50 ml centrifuge tube. Keep the digestion solution in 37 &#176;C water bath until use. 
	Note: The amount of the digestion solution prepared is sufficient for up to five adult mouse thymi, which can be incubated together in one 5 ml polystyrene round-bottom tube.</li>
	<li>Transfer the thymus to a 60 mm tissue culture dish containing 5-6 ml RPMI-1640.</li>
	<li>Dissect the thymus with 28 G insulin syringe needle into small pieces (about 1 mm3 square) to let the lymphocytes flow out.</li>
	<li>Rinse the thymic pieces with RPMI-1640 that is in the petri dish with glass Pasteur pipette. Tilt the dish and let the thymic fragments settle to the bottom. Slowly aspirate and discard the RPMI-1640 supernatant using a glass pipette. Repeat this rinsing step with 5-6 ml RPMI-1640 for 4-5 times with glass pipette until the solution is less opaque. 
	Note: Glass pipettes are recommended at this step, since thymic fragments tend to stick to plastic, resulting in excessive loss of thymic tissues. For the rest of the procedure, use plastic pipettes.</li>
	<li>After the final rinse, discard the supernatant as described in step 1.5. Add 3 ml digestion solution and transfer the thymus pieces and the solution into a 5 ml round-bottom polystyrene tube.</li>
	<li>Place the tube on a tube rotator and incubate at 37 &#176;C for 6 min at a rotation speed of 12 rpm.</li>
	<li>After incubation, let the thymic fragments settle to the bottom of the tube by gravity. Collect the supernatant with Pasteur pipette and transfer into a 50 ml centrifuge tube with 10 ml of washing solution. Centrifuge at 300 x g for 5 min, discard the supernatant and resuspend the cells in 10 ml washing solution. Keep on ice.</li>
	<li>Repeat steps 1.6-1.8 twice on the thymic fragments in the 5 ml round-bottom polystyrene tube. Pool the supernatant in the same 50 ml tube. 
	Note: Centrifugation is not necessary for the second and the third wash.</li>
	<li>After the final digestion, pipet up and down with 2 ml plastic serological pipette to break down any remaining fragments in the tube and transfer to the 50 ml centrifuge tube.</li>
	<li>Centrifuge the 50 ml tube at 300 x g for 5 min, aspirate the supernatant, resuspend in 10 ml of washing buffer and filter through 100 &#181;m cell strainer. Keep on ice.</li>
</ol>
2. Enrichment of TECs with Discontinuous Density Gradient
<ol>
	<li>Prepare 21% density gradient solution by mixing 2.4 ml density gradient medium (60% w/v of iodixanol in water) and 9.6 ml washing solution.</li>
	<li>Centrifuge the dissociated thymic cells in the 50 ml collection tube from step 1.11 at 300 x g for 5 min and resuspend in 2.5 ml washing buffer.</li>
	<li>Add 20 ml RPMI-1640 medium to the cell suspension.</li>
	<li>Fill 12 ml of 21% density gradient solution in a 10 ml serological pipet with an electronic pipettor. Place the tip of the serological pipet at the bottom of the 50 ml centrifuge tube containing cells, and allow the density gradient solution to drain to the bottom of the tube via gravity.
	<ol>
		<li>Gently expel the density gradient solution from the pipet to finish generating the two layers of different densities.</li>
	</ol>
	</li>
	<li>Centrifuge at 600 x g for 20 min at RT in a swinging-bucket rotor. Set the deceleration rate at the slowest level.</li>
	<li>Transfer the top layer and the interface to a new 50 ml tube. 
	Note: Most of the TECs are retained in the interface. Lymphocytes will precipitate to the bottom of the tube after centrifugation. If these cells are to be used for other purposes, rinse the pellet with 20 ml washing solution for three times. Resuspend the cells in 10 ml of washing buffer.</li>
	<li>Add washing solution up to 40 ml to the tube in step 2.6 and centrifuge at 400 x g for 6 min at 4 &#176;C. To remove the supernatant, aspirate with a pipet.</li>
	<li>Repeat the washing and the aspiration 2 more times as described in step 2.7.</li>
	<li>Resuspend in 2 ml of washing solution and count the cells with a hemocytometer.</li>
</ol>
3. Isolating TECs with FACS
<ol>
	<li>Adjust cells from step 2.9 at the concentration of 1x106 cells/100 &#181;l with washing solution and transfer the cells to a 5 ml round-bottom tube (polypropylene or polystyrene, as recommended by the instructions of the flow sorter).</li>
	<li>Add 2 &#181;l anti-Fc receptor IgG per 1x106 cells and incubate at 4 &#176;C for 10 min.</li>
	<li>Add anti-CD45 (1:150 dilution, 10 &#181;l per sample) and anti-EpCAM (1:100 dilution, 10 &#181;l per sample) antibodies and incubate at 4 &#176;C for more than 20 min.</li>
	<li>Wash the cells with 2 ml washing solution and centrifuge at 400 x g for 6 min at 4 &#176;C. Aspirate the supernatant with a pipette.</li>
	<li>Resuspend the cells in 1 ml 1x phosphate-buffered saline (PBS).</li>
	<li>Filter through 100 &#181;m strainer.</li>
	<li>Apply the cells to the sorting instrument. Select the lymphocyte and TEC population excluding the smallest cells on side-scattered light (SSC) / forward-scattered light (FSC) panel (dead cells), then gate on the selected population for CD45- EpCAM+ for sorting13.</li>
</ol>
4. Generation of TEC/EAK Aggregates
Note: Perform reagent preparation and steps involving cell mixing under the laminar hood.
<ol>
	<li>Prepare pA/G adaptor complexes by mixing 4 &#181;l of anti-His-tag IgG, 8 &#181;l of anti-EpCAM IgG and 2.6 &#181;l of pA/G in a sterile 1.5 ml microcentrifuge tube, and incubate at RT for 5 min.</li>
	<li>Adjust the sorted TECs from step 3.7 to 5x104 cells/ml with washing solution, and pipet 100 &#181;l to one well of a low evaporative, 96-well round-bottom tissue culture plate. Add 14.6 &#181;l pA/G adaptor complexes to the cells. Place the plate on a rocking platform for 20 min at 4 &#176;C.</li>
	<li>Wash once with 200 &#181;l washing solution. Centrifuge the plate at 400 x g for 6 min. Discard the supernatant with a micropipette.</li>
	<li>Wash once with 200 &#181;l 10% sucrose solution. Centrifuge at 400 x g for 6 min. Discard the supernatant with a micropipette.</li>
	<li>Resuspend cells in 30 &#181;l 10% sterile sucrose solution per well.</li>
	<li>Add 10 &#181;l of EAKIIH6 (7.5 mg/ml) and incubate for 5 min at RT.</li>
	<li>Add 20 &#181;l of EAK16-II (10 mg/ml) to the TEC mixture.</li>
	<li>Add 100 &#181;l of complete medium to the system to initiate gelation. Place the plate on a platform shaker for 15-20 min at RT.</li>
	<li>Place the plate in the incubator at 37 &#176;C and 5% CO2. After 2-4 hr, add another 100 &#181;l of complete medium.</li>
	<li>Change medium every other day until use.
	<ol>
		<li>Aspirate 100 &#181;l of the medium from the surface without disturbing the gel using a micropipette and gently add 100 &#181;l of pre-warmed medium by placing the pipette tip to the wall of the well.</li>
	</ol>
	</li>
</ol>
5. Characterization of the TEC/EAK Aggregates
<ol>
	<li>To examine the function of the TEC/EAK aggregates in vivo, collect the TEC/EAK hydrogel after O/N culture and transplant under the kidney capsule of an athymic nude mouse, using previously described procedure 11,14.</li>
	<li>Monitor the development of T cells in the periphery by harvesting 50-100 &#181;l blood samples into the EDTA-containing blood collection tube from the nude mice 4-6 weeks post transplantation and stain with a cocktail of anti-CD4, -CD8, -CD45, and -CD3 antibodies12.</li>
	<li>Analyze the percentages of T-lymphocytes in the peripheral blood leukocytes with flow cytometry (FCM), using single cell analysis software12.</li>
</ol>

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Authors declare no conflict of interest.

While TECs are the predominant population of the thymic stroma and play essential roles for the structure and function of the thymus glands, they represent only about 0.1-0.5% of the total thymic cellularity. They are also fragile cells as high percentages of cell death are occasionally observed following collagenase digestion, i.e., the treatment to dissociate TECs from the extracellular matrix (ECM). Their rarity (~200,000 per mouse thymus) and fragility made it a challenging task to isolate TECs. The recent u...

The thymus is the primary lymphoid organ responsible for the generation of a diverse population of pathogen-reactive, self-tolerant T cells that is essential to the function of the acquired immune system. It is a dynamic organ where developing thymocytes, immigrated from the bone marrow as lymphocyte progenitor cells, migrate through the sponge-like three-dimensional (3-D) matrix of the thymic stroma, undergo lineage-specification and differentiation, and eventually emigrate as mature T-cells. The success of this well programmed process depends largely on the cross-talk between the migrating thymocytes and the residential thymic epithelial cells (TECs), the predominant population of the thymic stroma that are essential for establishing and maintaining the integrity of the thymus microenvironment.
Based on their anatomical location and unique function, TECs can be divided into two subsets: the TECs in the cortex (cTECs) that are responsible for selecting self-MHC (major histocompatibility complex) restricted T-cells (positive selection), and the TECs in the medulla (mTECs) that are essential for eliminating autoreactive T-cells (negative selection) 1,2. Many factors (e.g., aging, infection, irradiation, drug treatments) can cause irreversible damages to the thymic epithelium, resulting in compromised adaptive immunity. Despite numerous attempts, restoring the thymic function has been challenging due to the difficulty to reproduce the thymic microenvironment. Notably, thymic three-dimensional (3-D) configuration is critical to the survival and function of TECs, whereas TECs cultured in a 2-D environment rapidly decrease the expression of genes critical for thymopoiesis 3,4.
EAK16-II (AEAEKAKAEAEAKAK) and its C-terminal histidinylated analogue EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) are low-molecular weight, amphiphilic oligopeptides that are soluble in deionized water, but undergo gelation to form &#946;-fibrils when exposed to ionic strength higher than 20 mM NaCl (normal salt concentration in human body fluid is 154 mM). This environmental responsive property makes them versatile building blocks to form 3D structures. The His-tag on EAKII-H6 provides a docking mechanism, by which the anti-His IgGs/Fc-binding recombinant protein A/G (&#945;H6:pA/G) complexes can serve as an adaptor to anchor protein drugs and other biomolecules (e.g., antibodies) on the hydrogel composite 5-8.
We have previously demonstrated that fluorescent-labeled IgG molecules anchored on the hydrogel can be retained at the injection site for up to 13 days 9. Furthermore, when the &#945;H6:pA/G adaptors anchored with anti-CD4 IgGs were added to the EAK16-II/EAKIIH6 (EAK) hydrogel, CD4+ T cells were specifically captured 10. Using similar technique, we have recently demonstrated that 3-D aggregation of TECs could be promoted in EAK hydrogel with adaptor complexes carrying TEC-specific anti-EpCAM antibodies (&#945;H6:pA/G:&#945;EpCAM). When transplanted underneath the kidney capsules of nude mice, the TEC clusters embedded in EAK hydrogel can effectively support the development of functional T cells in vivo11,12.
Here we illustrate our method to quickly and effectively purify TECs with fluorescence-activated cell sorting (FACS) and generate 3-D TEC aggregates with the EAK hydrogel system.

<table border="0" cellpadding="0" cellspacing="0" width="722">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1. TEC Isolation</td>
			<td style="width:175px;"></td>
			<td style="width:111px;"></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701(1 Gallon)</td>
			<td style="width:223px;">Ethanol 200 Proof, deionized water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Dissecting scissors, straight</td>
			<td>Fine Science Tools, Inc.</td>
			<td>91460-11</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Graefe forceps, straight</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11053-10</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Graefe forceps, curved</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11052-10</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">Washing solution</td>
			<td></td>
			<td></td>
			<td style="width:223px;">1X PBS, 0.1% BSA, 2mM EDTA</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">1x PBS (Phosphate buffered saline)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">BSA (Bovine serum albumin)</td>
			<td>Sigma</td>
			<td>A1470-100G</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">EDTA (ethylenediaminetetraacetic acid)</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:216px;">Digestion solution</td>
			<td></td>
			<td></td>
			<td style="width:223px;">9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">RPMI-1640</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Liberase TM Research Grade</td>
			<td>Roche</td>
			<td>05 401 127 001</td>
			<td style="width:223px;">referred as &#34;purified collagenase&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1M HEPES</td>
			<td>Lonza</td>
			<td>17-737E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Dnase I</td>
			<td>Roche</td>
			<td>10 104 159 001</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">50mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">60mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353002</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">1/2cc U-100 Insulin syringe 28G1/2</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">5mL Polystyrene round-bottom tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">5ml glass pipet</td>
			<td>Fisher Healthcare</td>
			<td>13-678-27E</td>
			<td style="width:223px;">Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">MACSmix tube rotator</td>
			<td>Miltenyi</td>
			<td>130-090-753</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">100um Cell strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Density gradient medium: OptiPrep</td>
			<td>Axis-Shield</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:223px;">Comments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">2. Cell sorting</td>
			<td></td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:216px;">5mL Polypropylene round-bottom tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD16/CD32 (Fc Block)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td style="width:223px;">Use as undiluted, 2uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD45-PercpCy5.5</td>
			<td>eBioscience</td>
			<td>45-0451-80</td>
			<td style="width:223px;">Use at 1:150, 10uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD326 (EpCAM)-PE</td>
			<td>eBioscience</td>
			<td>12-5791-82</td>
			<td style="width:223px;">Use at 1:100, 10uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">BD Influx</td>
			<td>BD Biosciences</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Single cell analysis software</td>
			<td>FlowJo</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:223px;">Comments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">2. EAK gel assembly</td>
			<td></td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-His-Tag</td>
			<td>AnaSpec</td>
			<td>29673</td>
			<td style="width:223px;">&#34;anti-His-Tag IgG&#34;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Purified anti-mouse CD326 (EpCAM)</td>
			<td>BioLegend</td>
			<td>118202</td>
			<td style="width:223px;">&#34;anti-EpCAM IgG&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Recombinant protein A/G</td>
			<td>Pierce Biotechnology</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:216px;">1.5mL Safe-Lock Tubes, Biopur, Sterile</td>
			<td>Fisher Healthcare</td>
			<td>05-402-24B</td>
			<td style="width:223px;">referred as &#34;1.5mL microcentrifuge tube&#34;&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">96-well, Tissue culture plate, Round-bottom with low evaporation lid</td>
			<td>BD Falcon</td>
			<td>353917</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rocking platform: Nutator Mixer no.1105</td>
			<td>BD Clay Adams</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">10% sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td style="width:223px;">Prepare with sterile distilled water</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2)</td>
			<td>American Peptide Company&#160;</td>
			<td></td>
			<td style="width:223px;">custom synthesized, 10mg/mL</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:216px;">EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2)</td>
			<td>American Peptide Company&#160;</td>
			<td></td>
			<td style="width:223px;">custom synthesized, 7.5mg/mL</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">Complete medium</td>
			<td></td>
			<td></td>
			<td style="width:223px;">RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">RPMI-1640&#160;</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">FBS (Fetal Bovine Serum)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td style="width:223px;">Heat inactivated before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">L-glutamine 200mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">NEAA (non-essential amino acid) 100x</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1M HEPES</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">2-Mercaptoethanol (100X)</td>
			<td>Millipore</td>
			<td>ES-007-E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Platform shaker: The Belly Dancer</td>
			<td>Stovall Life Sciences Inc.</td>
			<td>model: USBDbo</td>
			<td style="width:223px;"></td>
		</tr>
	</tbody>
</table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

To examine the effectiveness of using the density gradient separation protocol to enrich the CD45- stromal cells, cells harvested from both the interface and the precipitated lymphocyte pellets were stained with anti-CD45 and anti-EpCAM antibodies. Both anti-Ulex Europaeus Agglutinin 1 (UEA1) and anti-MHC Class II antibodies were also included in the staining cocktail to further identify the cTEC and mTEC subsets. As shown in Figure 1, we were able to routinely achieve cl...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.5K Views.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Video: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

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Chapters in this video

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Preparation and Applications of Organotypic Thymic Slice Cultures