Hirschil Trust, Michael Saperstein Medical Scholars Research Funds, and the NYU Whitehead fellowship supported this work.

1. Coating the Microplate Wells
Note: The goal of this step is to coat polystyrene surfaces with ICAM-1 to serve as ligands for T cell LFA-1.
<ol>
	<li>Prepare the following solutions:
	<ol>
		<li>Prepare the coating solution by resuspending recombinant ICAM-1 with phosphate buffered saline (PBS) solution (137 mM NaCl,&#160;2.7 mM KCl,&#160;10 mM Na2HPO4,&#160;2 mM KH2PO4,&#160;pH 7.4) enriched with calcium (1 mM CaCl2) and magnesium (2 nM MgCl2). Make sure that the final concentration of ICAM-1 is 10 &#956;g/ml, made from a 0.7 mg/ml stock solution and kept in a -80 &#176;C freezer.</li>
		<li>Prepare the adhesion solution by enriching PBS with 0.5% human (or bovine) serum albumin (HSA/BSA), 2 mM MgCl2, and 1 mM CaCl2.</li>
		<li>Prepare the wash solution by adding 2 mM MgCl2 and 1 mM CaCl2 to PBS. Warm all solutions to 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Wash 24 wells with 50 &#956;l of wash solution. This will include unwashed wells, positive (PMA treated cells), and negative (uncoated wells) controls. For each condition set at least three wells (triplicate).</li>
	<li>Add 50 &#956;l of coating solution to each well. Do not add to the uncoated control wells. Incubate for one hour inside 37 &#176;C incubator.</li>
	<li>Aspirate the coating solution gently, without allowing the tip to touch the bottom of the wells. Wash once with 50 &#956;l of wash solution.</li>
	<li>Add 50 &#956;l of adhesion solution and incubate the microplate for one hr&#160;inside 37 &#176;C incubator.</li>
	<li>Aspirate gently and wash once with 50 &#956;l wash solution. Use the plate on the same day, however it is possible to keep it at 4 &#176;C over night, and use it the following day.</li>
</ol>
2. T Cell Isolation from Blood
<ol>
	<li>Add human T cell enrichment cocktail at 50 &#956;l/ml of whole blood (e.g. for 3 ml of anticoagulant (heparin, EDTA, or citrate) whole blood, add 150 &#956;l of cocktail). Mix well.</li>
	<li>Incubate 20 min&#160;at room temperature.</li>
	<li>To a 50&#160;ml centrifuge tube add 3 ml of treated blood and an equal volume of PBS (without calcium and magnesium) enriched with 1% D-glucose at room temperature. Mix gently with a Pasteur pipette.</li>
	<li>Add 15&#160;ml Ficoll-Paque PLUS to the centrifuge tube.</li>
	<li>Carefully layer the 6 ml diluted blood sample on the lymphocyte isolation medium.</li>
	<li>Centrifuge at 400 x g for 20 min&#160;at 20 &#176;C with break off.</li>
	<li>Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface. The upper layer of plasma may be saved for later use. Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube.</li>
	<li>Add 3 volumes (9 ml) of PBS to the lymphocytes. Suspend the cells by gently drawing them in and out of a Pasteur pipette.</li>
	<li>Centrifuge at 400 x g for 5 min&#160;at 20 &#176;C.</li>
	<li>Remove the supernatant. The lymphocytes should now be suspended in the medium appropriate to the application. Transfer the cells to a T-75 culture flask in 20 ml RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin.</li>
</ol>
3. Preparation of the Cells
Note: A prerequisite is for the cells to be labeled with a fluorescent reagent. In this protocol use carboxy fluorescein succinimidyl ester (CFSE), however green fluorescent protein (GFP) expressing cells are an acceptable alternative.
<ol>
	<li>Count 2.4 x 106 T cells (1 x 105 for each well) using a hemocytometer.</li>
	<li>Resuspend the cells in culture media lacking serum (serum starvation). Incubate the cells for 2&#160;hr&#160;in the 37 &#176;C incubator.</li>
	<li>Centrifuge the cells for 5 min&#160;(400 x g). Aspirate the media and resuspend in 1 ml PBS.</li>
	<li>Add CFSE at 1:1,000 dilution (stock made to 5 mM). Cover from light and incubate for 8 min&#160;at room temperature.</li>
	<li>Stop the reaction by adding 10 ml of 37 &#176;C PBS and spin at 400 x g for 5 min.</li>
	<li>Re-suspend cell pellet with 1.2 ml of pre-warmed adhesion solution (1 x 105 cells per 50 &#956;l).</li>
</ol>
4. Stimulation of the Cells
Note: In order to initiate adhesion, cells must be stimulated. In the following experiment the cells are stimulated via the TCR using anti-CD3 crosslinking antibodies, however soluble SDF-1 could serve as an alternative. Depending on the goal of the experiments, various pharmacological reagents could be added before or during stimulation to study the impact on cellular adhesion. Phorbol myristate acetate (PMA) is a phorbol ester that is structurally similar to the second messenger diacyl glycerol (DAG) and therefore activates multiple kinases downstream the TCR (mainly Protein kinase C); here it is used as a positive control.
<ol>
	<li>Warm the microplate to 37 &#176;C.</li>
	<li>Divide the cells into 8 empty 1.5 ml tubes (150 &#956;l in each tube), one tube for each condition.</li>
	<li>Treat the cells accordingly:
	<ol>
		<li>Stimulate one tube with PMA at 10 ng/ml to serve as positive control. Keep three tubes untreated, to serve as control for stimulation (&#8220;no stimulation&#8221;), unwashed loading control (&#8220;no wash&#8221;), and control for ICAM-1 coating (&#8220;uncoated&#8221;).</li>
		<li>Stimulate four tubes with different concentrations of anti-CD3 antibodies (0.1, 1, 5, and 10 &#956;g/ml). Continue to the next step without delays.</li>
	</ol>
	</li>
	<li>Aliquot 50 &#956;l of stimulated cell mix into each empty well. The final number of cells per well is 1 x 105.</li>
	<li>Place the microplate in the 37 &#176;C incubator for 15 min. 
	Note: Some T cell lines require shorter stimulation time. During this time, the cells will settle down to the bottom of the wells and adhere to the target ligands.</li>
</ol>
5. Washing Away Non-adherent Cells
Note: The goal of this step is to remove cells that were not able to form tight contacts with the ligand-coated surfaces. Use a multichannel pipette for this step in order to ensure that the same physical forces are applied to all the wells. It is important to keep the indicated control wells unwashed to assist in the calculation of the percentage of adherent cells.
<ol>
	<li>Add 150 &#956;l of warm adhesion solution to each wells. Shake the plate gently for few seconds before removing the medium from the wells. Do not touch the bottom of the wells with the tips.</li>
	<li>Repeat this step three times. Change the direction of the pipette when pipetting the buffer in and out.</li>
</ol>
6. Determining the Percentage of Adherent Cells
Note: In this step a fluorescent plate reader is used to measure the fluorescent intensity within each well. The intensity is in direct correlation with the number of the adherent cells.
<ol>
	<li>Turn on the plate reader. Open the plate reader software by clicking the shortcut icon on the desktop.
	<ol>
		<li>From the &#8220;Task&#8221; menu click &#8220;New&#8221; to set up a new protocol.</li>
		<li>From the &#8220;Actions&#8221; menu choose the &#8220;Read&#8221; option. Click &#8220;Fluorescent intensity&#8221; and hit the &#8220;Ok&#8221; tab.</li>
		<li>From the drop down menu select &#8220;Excitation wavelength 485&#8221; and &#8220;Emission wavelength 528&#8221;. Hit the &#8220;Ok&#8221; tab.</li>
		<li>In &#8220;Optic&#8221; option menu select &#8220;Bottom&#8221; and hit the &#8220;Ok&#8221; tab. Go back to the &#8220;Actions&#8221; menu and set the temperature to 37 &#176;C and click &#8220;Ok&#8221;.</li>
		<li>Insert the microplate into the plate reader and click &#8220;Run&#8221;. The result will be exported into excel spreadsheet.</li>
	</ol>
	</li>
	<li>Calculate the percentage of adherent cells using the following formula: Percentage of adherent cells = (average fluorescent intensity read in the washed wells)/(average fluorescent intensity read in the unwashed cells) x 100%.</li>
</ol>

<ol>
	<li>Dustin, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+as+messengers+in+T+cell+adhesion+signaling.">Membranes as messengers in T cell adhesion signaling.</a> Nat Immunol. 5 (4), 363-372 (2004).</li><li>Mor, 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=GTPases+and+LFA-1+reciprocally+modulate+adhesion+and+signaling.">GTPases and LFA-1 reciprocally modulate adhesion and signaling.</a> Immunol Rev. 218, 114-125 (2007).</li><li>Rose, D. 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=Integrin+modulation+and+signaling+in+leukocyte+adhesion+and+migration.">Integrin modulation and signaling in leukocyte adhesion and migration.</a> Immunol Rev. 218, 126-134 (2007).</li><li>Alon, R., Feigelson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-triggered+leukocyte+arrest:+force-regulated+bi-directional+integrin+activation+in+quantal+adhesive+contacts.">Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts.</a> Curr Opin Cell Biol. 24 (5), 670-676 (2012).</li><li>Griffith, J. W., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cells+in+motion:+migrating+toward+improved+therapies.">Targeting cells in motion: migrating toward improved therapies.</a> Eur J Immunol. 43 (6), 1430-1435 (2013).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecules+and+actin+cytoskeleton+at+immune+synapses+and+kinapses.">Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.</a> Curr Opin Cell Biol. 19 (5), 529-533 (2007).</li><li>Springer, T. A., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inside-out+signaling+and+the+immunological+synapse.">Integrin inside-out signaling and the immunological synapse.</a> Curr Opin Cell Biol. 24 (1), 107-115 (2012).</li><li>Mor, 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=D1+regulates+lymphocyte+adhesion+via+upregulation+of+Rap1+at+the+plasma+membrane.">D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.</a> Mol Cell Biol. 29 (12), 3297-3306 (2009).</li><li>Bivona, T. G., 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=Rap1+up-regulation+and+activation+on+plasma+membrane+regulates+T+cell+adhesion.">Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion.</a> J Cell Biol. 164 (3), 461-470 (2004).</li><li>Zeltzer, E., 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=Diminished+adhesion+of+CD4++T+cells+from+dialysis+patients+to+extracellular+matrix+and+its+components+fibronectin+and+laminin.">Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin.</a> Nephrol Dial Transplant. 12 (12), 2618-2622 (1997).</li><li>Bhattacharyya, S. P., 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=Both+adhesion+to+immobilized+vitronectin+and+Fc+epsilon+RI+cross-linking+cause+enhanced+focal+adhesion+kinase+phosphorylation+in+murine+mast+cells.">Both adhesion to immobilized vitronectin and Fc epsilon RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.</a> Immunology. 98 (3), 357-362 (1999).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li></ol>

The authors have nothing to disclose.

There are several assays to study signals to LFA-1 activation and T cell adhesion12. Flow cytometry is used to measure LFA-1 affinity states in living cells using monoclonal antibodies that bind selectively to either bended or extended LFA-1. One of the limitations of this method is that it does not take into account the integrins&#8217; avidity. Migration assays are useful tools but they measure migration, and not measly adhesion at a specific time point. Mouse models to study T lymphocyte migration (e.g....

T lymphocyte adhesion is a fundamental process in the immune response1. It is required for T cell interaction with endothelial cells decorating the capillary walls, for scanning antigen presenting cells (APC) within lymph nodes, and for the formation of immunological synapses (IS) with target cells2. These requirements are functionally and kinetically distinct. The process of lymphocytes extravasation consists of chemoattraction, rolling, firm adhesion, and transmigration. The transition from rolling to firm adhesion requires T cells to respond to a G protein coupled receptor signal rapidly. This response yields an integrin ligand interaction that slows and arrests the rolling cell3. An immediate change in integrin avidity mediates this process. Migration requires dynamic interactions betweenTcells and endothelial cells with the formation of adhesions at the &#39;front end&#39; and breakage of adhesions at the &#39;rear end&#39; with an interval of about a minute between forming and breaking4. The IS forms inminutes, but needs to remain intact for hours6.
Interestingly, one adhesion molecule, the integrin family member lymphocyte function associated antigen (LFA)- 1, is essential for all these processes5. LFA-1 mediates adhesion through interactions with several members of the immunoglobulin superfamily. The most extensively studied ligand with the highest affinity to LFA-1 is the intercellular adhesion molecule (ICAM)-1. Non-activated circulating lymphocytes express low affinity LFA-1 on the cell surface, and therefore are unable to adhere to ICAM-1-coated surfaces. LFA-1 affinity is variable and regulated by several signaling events such as G protein coupled receptor activation, cytokine stimulation, and signals mediated by T cell receptors (TCR).The resulting high affinity form of LFA-1 conveys intracellular activation to the extracellular space through interactions with ICAM-1. This pathway is termed inside-out signaling7. Likewise, signaling through LFA-1 from the extracellular space is called outside-in signaling.
The intracellular signaling cascades involved in inside-out and outside-in signaling are a major focus of current research. The small GTPase Rap1 has recently emerged as the key component of inside-out signaling that is common to both TCR ligation and cytokine signaling8. The critical role of Rap1 in integrin activation is highlighted by the discovery that overexpression of Rap1 stimulates integrin-dependent adhesion of T cells, whereas T cell adhesion is blocked by expression of dominant negative Rap19. These advances in our understanding of integrin regulation by Rap1 have been accomplished using in&#160;vitro tools. Among them is the static adhesion assay described here.
The overall goal of this method is to study T cell adhesiveness to ICAM-1 coated surfaces. More specifically, it is used to objectively measure and quantify LFA-1 affinity toward its counter ligands in live cells at real time, under different conditions. This technique uses polystyrene wells coated with ICAM-1 to mimic the cellular surfaces that the T cells interact with. Many previously described static T cell adhesion assays were experimentally complex. These assays often required for T cells to be radioactively labeled, utilized cultured bovine corneal cells to create an extracellular matrix as the substrate for T cell adhesion, or called for non-physiological T cell stimulation over an extended duration to promote T cell adhesion10. The use of fluorometric measurement to quantify T cells following the adhesion incubation is a more sensitive and accurate method of quantification as compared to flow cytometry and microscopy, as are utilized in many other assay systems11. Additionally, single cell microscopic analysis of integrin localization does not allow for the broad, population based analysis in the same way as the fluorometric measurement. While activation state-specific LFA-1 antibodies are commercially available, these antibodies offer low sensitivity relative to the method outlined here. The main advantage over alternative techniques is its simplicity and the ability to examine multiple experimental conditions simultaneously. When considering this method for a specific application, one should take into account that T cells should be negatively selected, freshly isolated, and labeled with a fluorescent marker.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Plate reader</td>
			<td style="width:160px;">BioTek&#160;</td>
			<td style="width:136px;">Synergy H1 Hybrid</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Multichannel pipette</td>
			<td style="width:160px;">Fisher</td>
			<td style="width:136px;">21-377-829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">96-well microplate with optical bottom</td>
			<td style="width:160px;">Corning Costar</td>
			<td style="width:136px;">3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Recombinant ICAM-1</td>
			<td style="width:160px;">R&#38;D Systems</td>
			<td style="width:136px;">ADP4-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Anti-CD3 antibodies</td>
			<td style="width:160px;">Ancell</td>
			<td style="width:136px;">144-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">PMA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">79346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">BSA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">CFSE</td>
			<td style="width:160px;">Molecular Probes</td>
			<td style="width:136px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">RPMI</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">DPBS containing calcium and magnesium</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Peripheral lymphocytes</td>
			<td style="width:160px;">e.g. mouse or human</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Open Gen 5 software</td>
			<td>BioTek</td>
			<td>Version 2.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 and 50 mL centrifuge tubes</td>
			<td>Fisher</td>
			<td>352099, 352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-75 culture flasks</td>
			<td>Fisher</td>
			<td>50-754-1366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RosetteSep T cell enricment kit</td>
			<td>StemCell Technologies</td>
			<td>15061</td>
			<td></td>
		</tr>
	</tbody>
</table>

static adhesion assay for the study of integrin activation in t lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Below is an example of adhesion assay using primary T cells stimulated with various concentrations of anti-CD3 antibodies. It is useful to know the fluorescence of unwashed cells (i.e. total loading). Unstimulated cells serve as a negative control and PMA treated cells are the positive control for anti-CD3 antibodies. Cells plated in uncoated wells serve as a control for ICAM-1. In a typical experiment the percentage of adherent PMA treated cells is between 40% to 50% while the percentage of unstimulated cells i...

Watch this Scientific Journal Video about Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes at JoVE.com

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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Unit 1

Cell-Matrix Interactions

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15.9K visualizações.  New York University School of Medicine.  Ensaio de adesão estático é uma ferramenta poderosa que pode ser utilizado para modelar as interacções entre linfócitos T e de outros tipos de células. Interacções são geradas por injecção de células T marcadas em poços revestidos com moléculas de adesão, enquanto que um leitor de placa é utilizada para quantificar o número de células aderentes a seguir lavagens em série. 

Vídeo: Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...