Name: promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel
Uploaded: 2016-06-27T14:00:00
Description: 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

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 i...

<ol>
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O., Levi, D., Chentoufi, A. A., Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+proinsulin-producing+medullary+thymic+epithelial+cell+clones.">Isolation and characterization of proinsulin-producing medullary thymic epithelial cell clones.</a> Diabetes. 55, 2595-2601 (2006).</li><li>Williams, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+complex+thymus+stromal+cell+populations:+rapid+thymic+epithelia+preparation+characterizes+radiation+injury.">Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury.</a> Clin Transl Sci. 2, 279-285 (2009).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+identification,+and+purification+of+murine+thymic+epithelial+cells.">Isolation, identification, and purification of murine thymic epithelial cells.</a> J Vis Exp. , e51780 (2014).</li><li>Graham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+monocytes+from+whole+human+blood.">Separation of monocytes from whole human blood.</a> ScientificWorldJournal. 2, 1540-1543 (2002).</li><li>Li, X., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+subcellular+membrane+vesicles+of+epithelial+and+nonepithelial+cells+by+OptiPrep+density+gradient+ultracentrifugation.">Fractionation of subcellular membrane vesicles of epithelial and nonepithelial cells by OptiPrep density gradient ultracentrifugation.</a> Methods Mol Biol. 440, 97-110 (2008).</li><li>Mita, 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=Purification+method+using+iodixanol+(OptiPrep)-based+density+gradient+significantly+reduces+cytokine+chemokine+production+from+human+islet+preparations,+leading+to+prolonged+beta-cell+survival+during+pretransplantation+culture.">Purification method using iodixanol (OptiPrep)-based density gradient significantly reduces cytokine chemokine production from human islet preparations, leading to prolonged beta-cell survival during pretransplantation culture.</a> Transplant Proc. 41, 314-315 (2009).</li><li>Anderson, G., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+epithelial+cells:+working+class+heroes+for+T+cell+development+and+repertoire+selection.">Thymic epithelial cells: working class heroes for T cell development and repertoire selection.</a> Trends Immunol. 33, 256-263 (2012).</li><li>Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+central+tolerance.">A central role for central tolerance.</a> Annu Rev Immunol. 24, 571-606 (2006).</li><li>St-Pierre, C., 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=Transcriptome+sequencing+of+neonatal+thymic+epithelial+cells.">Transcriptome sequencing of neonatal thymic epithelial cells.</a> Sci Rep. 3, 1860 (2013).</li></ol>

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 ...

The overall goal of this procedure is to demonstrate the use of the EAK 16-II/EAKIIH6 peptide in promoting thymic epithelial cell aggregation in a three-dimensional structure for supporting thymic epithelial self-survival and function. This method may help answer key questions in the thymus bioengineering field. The main advantages of this technique is that it utilizes EAK-16-II/EAKIIH6 peptide, promotes thymic epithelial cell aggregation, is biodegradable, and has a potential to be used for injections.To harvest the thymus, begin by placing a three to five week old C57 black 6J mouse in the supine position and wet the hair with 70%ethanol. Using sharp, straight dissecting scissors, make a small horizontal incision through the abdominal skin. Then pull the skin on both sides of the incision to expose the abdominal cavity.Next, make a horizontal cut just under the lowest rib and cut sideways to the diaphragm. Using forceps, pull the xiphoid process and cut through the middle of the ribs along both sides of the chest. Pull up the ribs and sternum so that the thymus is clearly visible.Then, using a curved forceps, gently scoop the lobes of the thymus off of the connective tissue and place them in washing solution. After the rinse, transfer the thymus into a 16 milliliter tissue culture dish containing five to six milliliters of RPMI 1640 and use a 28 gauge insulin needle to dissect the tissue into approximately one cubic millimeter square pieces. Next, use a glass pasteur pipette to rinse the tissue fragments with the medium in the dish.Then, tilt the dish, letting the fragments settle to the bottom. Slowly remove the medium supernatant by aspiration. Then repeat the wash four to five more times with five to six milliliters of fresh medium each time until the solution becomes less opaque.After the final wash, add three milliliters of digestion solution to the dish and transfer the thymus pieces and solution into a five milliliter round-bottom polystyrene tube. Incubate the tissues on a tube rotator at 37 degrees celsius for six minutes at 12 rpm. At the end of the rotation, let the fragments settle to the bottom of the tube and use a pasteur pipette to transfer the supernatant into a 15 milliliter conical tube containing 10 milliliters of washing solution.Centrifuge the supernatant and resuspend the cells in 10 milliliters of fresh washing solution on ice. Then digest the thymic fragments two more times in the rotator as just demonstrated, pulling the supernatant in the same 50 milliliter tube. After the final digestion, use a plastic two milliliter serological pipette to triturate any remaining fragments and transfer the tissues'flurry to the 50 milliliter tube.Spin down the tube to collect the cells and resuspend the pellet in 10 milliliters of washing buffer. Then, filter the cells through a 100 micron strainer while the tube is placed on ice. To enrich for the thymic epithelial cells, spin down the cells again and resuspend the pellet in 2.5 milliliters of washing buffer.Next, add 20 milliliters of RPMI 1640 to the cells and use a 10 milliliter serological pipette to carefully layer 12 milliliters of freshly prepared 21%density gradient solution under the cells by gravity. Now, separate the cells by centrifugation in a swinging bucket rotor at the slowest deceleration rate. At the end of the centrifugation, transfer the top layer and the interface into a new 50 milliliter tube and add up to 40 milliliters of washing solution to the cells.Remove the density gradient by three consecutive centrifugations using a pipette to aspirate the supernatant after each wash. Then resuspend the final pellet in two milliliters of washing solution for counting. To generate the thymic epithelial cell EAK aggregates, adjust the thymic epithelial cells to a five times 10 to the four cells per milliliter dilution in washing solution and seed 100 microliters of cells per well into a low evaporative 96 well, round-bottom, tissue culture plate.Next, add 14.6 microliters of freshly prepared protein AG adapter complexes to each well and place the plate on a rocking platform at four degrees celsius. After 20 minutes, wash the cells with 200 microliters of washing solution per well, using a micropipette to remove the supernatant. Then, wash the cells again with 200 microliters of 10%sucrose solution.Resuspend the pellets in 30 microliters of 10%sterile sucrose solution per well. Then, incubate the cells in 10 microliters of EAKIIH6 for five minutes at room temperature. At the end of the incubation, add 20 microliters of EAK 16-II, followed by 100 microliters of complete medium and place the plate on a platform at room temperature to initiate the gelation.After 15 to 20 minutes, transfer the plate to a 37 degrees celsius and 5%carbon dioxide incubator for two to four hours. Then add another 100 microliters of complete medium and return the plate to the incubator. To change the medium, use a micropipette to aspirate 100 microliters of the medium from the surface of the cultures without disturbing the gel and place the tip against the walls of each well to seed the cultures with 100 microliters of pre-warmed medium.Using the density gradient separation protocol as just demonstrated, CD45 negative stromal cells can routinely be enriched 15 to 20 times as illustrated in these representative dot plots. Here, thymic epithelial cell aggregates, prepared from four week old red fluorescent expressing transgenic mice as just demonstrated, were cultured for two days in vitro and examined by confocal microscopy. Once mastered, thymic epithelial cells can be enriched for flow sorting in three to four hours.After its development, this technique paved the way for researchers in the field of thymus tissue engineering to develop injectable, functional mini thymus units to modulate adaptive immune system.

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

Research

JoVE Journal

Immunology and Infection

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&amp;#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 ...

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 ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...