Name: spatial and temporal control of t cell activation using a photoactivatable agonist
Uploaded: 2018-04-25T13:00:00
Description: Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

We thank members of the Huse lab for advice and assistance. Supported by the US National Institutes of Health (R01-AI087644 to M.H. and P30-CA008748 to Memorial Sloan-Kettering Cancer Center).

1. Preparation of Stimulatory Glass Surfaces
<ol>
	<li>Coat eight-well chambered coverglass with biotinylated poly-L-lysine (Bio-PLL) diluted 1:500 in distilled, deionized water (ddH2O). Incubate for 30 min at room temperature (RT).</li>
	<li>Wash with H2O.</li>
	<li>Dry for 2 h at RT.</li>
	<li>Block Bio-PLL coated surfaces with blocking buffer (HEPES-buffered saline [10 mM HEPES pH 7.4, 150 mM NaCl], with 2% BSA) for 30 min at RT.
	<ol>
		<li>Dissolve 5 mg of poly-L-lysine hydrobromide in 1 mL o...

<ol>
	<li>Dustin, M. L., Chakraborty, A. K., Shaw, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+structure+and+function+of+the+immunological+synapse.">Understanding the structure and function of the immunological synapse.</a> Cold Spring Harb.Perspect.Biol. 2, a002311 (2010).</li><li>Liu, X., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Synapse+Formation:+Cell+Polarity+During+T+Cell-APC+Interaction.">Immunological Synapse Formation: Cell Polarity During T Cell-APC Interaction.</a> Cell Polarity 1. , 247-275 (2015).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+A+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: A role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Function+of+the+Immunological+Synapse+from+Studies+with+Supported+Planar+Bilayers.">Insights into Function of the Immunological Synapse from Studies with Supported Planar Bilayers.</a> Current topics in microbiology and immunology. 340, (2010).</li><li>Chauveau, A., Le Floc'h, A., Bantilan, N. S., Koretzky, G. a., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+kinase+&#945;+establishes+T+cell+polarity+by+shaping+diacylglycerol+accumulation+at+the+immunological+synapse.">Diacylglycerol kinase &#945; establishes T cell polarity by shaping diacylglycerol accumulation at the immunological synapse.</a> Sci. Signal. 7, ra82 (2014).</li><li>Huse, 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=Spatial+and+Temporal+Dynamics+of+T+Cell+Receptor+Signaling+with+a+Photoactivatable+Agonist.">Spatial and Temporal Dynamics of T Cell Receptor Signaling with a Photoactivatable Agonist.</a> Immunity. 27, 76-88 (2007).</li><li>Quann, E. J., Merino, E., Furuta, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+diacylglycerol+drives+the+polarization+of+the+microtubule-organizing+center+in+T+cells.">Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.</a> Nat Immunol. 10, 627-635 (2009).</li><li>Basu, R., Chen, Y., Quann, E. J., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variable+hinge+region+of+novel+PKCs+determines+localization+to+distinct+regions+of+the+immunological+synapse.">The variable hinge region of novel PKCs determines localization to distinct regions of the immunological synapse.</a> PLoS One. 9, 1-8 (2014).</li><li>Liu, X., Kapoor, T. M., Chen, J. K., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+promotes+centrosome+polarization+in+T+cells+via+reciprocal+localization+of+dynein+and+myosin+II.">Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 11976-11981 (2013).</li><li>Quann, E. J., Liu, X., Altan-Bonnet, G., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cascade+of+protein+kinase+C+isozymes+promotes+cytoskeletal+polarization+in+T+cells.">A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.</a> Nat. Immunol. 12, 647-654 (2011).</li><li>Boniface, J. J., Reich, Z., Lyons, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+T+cell+receptor+binding+to+peptide-MHC:+evidence+for+a+general+mechanism+of+molecular+scanning.">Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 11446-11451 (1999).</li><li>Garboczi, D. N., Hung, D. T., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-A2-peptide+complexes:+refolding+and+crystallization+of+molecules+expressed+in+Escherichia+coli+and+complexed+with+single+antigenic+peptides.">HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 3429-3433 (1992).</li><li>Huse, M., Lillemeier, B. F., Kuhns, M. S., Chen, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+use+two+directionally+distinct+pathways+for+cytokine+secretion.">T cells use two directionally distinct pathways for cytokine secretion.</a> Nat. Immunol. 7, 247-255 (2006).</li><li>Abeyweera, T. P., Merino, E., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+signaling+blocks+activating+receptor+clustering+and+induces+cytoskeletal+retraction+in+natural+killer+cells.">Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.</a> J. Cell Biol. 192, 675-690 (2011).</li><li>Nguyen Duc, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Generalizable+Platform+for+the+Photoactivation+of+Cell+Surface+Receptors.">A Generalizable Platform for the Photoactivation of Cell Surface Receptors.</a> ACS Chem. Biol. 10, 2435-2440 (2015).</li><li>Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+precise+in+vivo+control+of+intracellular+signalling.">Temporally precise in vivo control of intracellular signalling.</a> Nature. 458, 1025-1029 (2009).</li><li>Xu, Y., 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=Optogenetic+control+of+chemokine+receptor+signal+and+T-cell+migration.">Optogenetic control of chemokine receptor signal and T-cell migration.</a> Proc. Natl. Acad. Sci. 111, 6371-6376 (2014).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461, 997-1001 (2009).</li><li>Guntas, 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=Engineering+an+improved+light-induced+dimer+(iLID)+for+controlling+the+localization+and+activity+of+signaling+proteins.">Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.</a> Proc. Natl. Acad. Sci. 112, 112-117 (2015).</li><li>Wu, Y. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+photoactivatable+Rac+controls+the+motility+of+living+cells.">A genetically encoded photoactivatable Rac controls the motility of living cells.</a> Nature. 461, 104-108 (2009).</li><li>Pathak, G. P., Vrana, J. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+cell+function+using+engineered+photoreceptors.">Optogenetic control of cell function using engineered photoreceptors.</a> Biol. cell/under auspices Eur. Cell Biol. Organ. 105, 59-72 (2014).</li><li>Grusch, 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=Spatio-temporally+precise+activation+of+engineered+receptor+tyrosine+kinases+by+light.">Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.</a> EMBO J. 33, 1713-1726 (2014).</li><li>Yi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+repositioning+in+T+cells+is+biphasic+and+driven+by+microtubule+end-on+capture-shrinkage.">Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.</a> J. Cell Biol. 202, 779-792 (2013).</li></ol>

In recent years, light has emerged as an excellent tool for spatiotemporally controlled activation of cellular processes. Various methodologies have been developed, each with associated advantages and disadvantages. The system described here, which is based on the decaging of immobilized, extracellular ligands, is ideally suited for the analysis of rapid, subcellular, polarized signaling responses. This approach has been applied to examine IS formation in T cells as described above. Additionally, caged ligands for other ...

T lymphocytes (T cells) play a central role in cellular immunity by recognizing antigenic peptides displayed in the context of cell surface MHC. Antigen recognition, which is mediated by the TCR, drives the differentiation of na&#239;ve T cells and promotes the delivery of cytolytic and communicative responses by effector populations. TCR engagement also induces dramatic changes in cellular architecture. Within minutes, the T cells gloms onto the side of the antigen-presenting cell (APC), forming a polarized interface known as the immunological synapse (IS)1,2. The IS potentiates T cell effector responses by enabling the directional release of cytokines or, in the case of cytotoxic T lymphocytes (CTLs), lytic proteins that destroy the APC.
TCR engagement by pMHC induces the rapid phosphorylation of multiple downstream adaptor molecules, including Linker for the Activation of T cells (LAT), which ultimately promotes robust remodeling of the synaptic cytoskeleton2. Cortical filamentous actin (F-actin) drives T cell spreading over the APC surface, and then resolves into an annular structure characterized by F-actin accumulation at the IS periphery and depletion from the center. F-actin ring formation is tightly coupled to the reorientation of the microtubule organizing center (MTOC, also called the centrosome in T cells) to a position just beneath the center of the interface. Both events occur within minutes of initial antigen recognition and establish the architectural context in which subsequent activation events and effector responses occur.
To study IS formation, various labs have developed approaches in which the APC is replaced by a glass surface that either contains immobilized TCR ligands or supports a lipid bilayer that itself contains the ligands3,4. T cells form IS-like contacts on these surfaces that can be imaged by total internal reflection fluorescence microscope (TIRF) or confocal microscopy, enabling high-resolution studies of early T cell activation and IS formation.
Although these approaches have allowed for excellent visualization of the fully assembled IS, much of the signaling following TCR:pMHC ligation occurs within seconds, complicating efforts to determine the sequence of events following TCR activation accurately. To circumvent this issue, a photoactivation approach has been developed, in which photoactivatable pMHC is used to achieve spatiotemporal control of TCR activation5,6,7. In this system, T cells are attached to glass surfaces containing photoactivatable pMHC that is non-stimulatory to the TCR until irradiated with ultraviolet (UV) light. UV irradiation of a micron sized region of the surface beneath the T cell removes the photocage creating a stimulatory zone that can be recognized by the T cell. Subsequent signaling events and cytoskeletal remodeling are then monitored using genetically encoded fluorescent reporters. Two photoactivatable versions of antigenic peptides, moth cytochrome c88-103 (MCC) and ovalbumin257-264 (OVA), which are presented in the context of the class II MHC I-Ek and the class I MHC H2-Kb, respectively, have been developed (Figure 1). This enables the analysis of both CD4+ T cells specific for MCC- I-Ek (expressing the 5C.C7, 2B4, or AND TCRs) and CD8+ T cells specific for OVA-H2-Kb (expressing the OT1 TCR).
Over the past decade, the TCR photoactivation and imaging approach has been utilized to establish the precise kinetics of early TCR signaling steps and also to identify the molecular pathways governing polarized cytoskeletal remodeling5,6,7,8,9,10. For example, the assay was instrumental in determining that centrosome reorientation toward the APC is mediated by a localized gradient of the lipid second messenger diacylglycerol centered at the IS. It is anticipated that this methodology will continue to be valuable for applications that demand high-resolution imaging analysis of T cell function.

<table border="0" cellpadding="0" cellspacing="0" width="1152">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right" style="width:119px;">155361</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link NHS-Biotin</td>
			<td>Thermofischer Scientific</td>
			<td align="right">20217</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right">434301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BirA-500: BirA biotin-protein ligase standard reaction kit</td>
			<td>Avidity</td>
			<td>BirA500</td>
			<td>Will be used to biotinylate proteins</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated Hb I-EK&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference 6. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-MCC I-EK&#160;</td>
			<td>Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated &#945;H2-Kk antibody</td>
			<td style="width:211px;">BD Biosciences</td>
			<td align="right">553591</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-OVA H2-Kb</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated KAVY H2-Db&#160;</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated ICAM1&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Hand held UV lamp</td>
			<td style="width:211px;">UVP</td>
			<td style="width:119px;">UVGL-25</td>
			<td>Lamp is held &#60; 1 cm from the sample.&#160; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Olympus IX-81 OMAC TIRF system.</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Additional information about the imaging system can be found in Figure 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Mosaic digital diaphragm</td>
			<td style="width:211px;">Andor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Slidebook software</td>
			<td style="width:211px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial and temporal control of t cell activation using a photoactivatable agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

The photoactivation and imaging approach allows for observation and facile quantification of rapid, polarized signaling responses. To illustrate its capabilities, reproduced here is an experiment examining the spatiotemporal correlation between TCR-induced DAG accumulation and centrosome reorientation. 5C.C7 T cell blasts were retrovirally transduced with two fluorescent reporters: a DAG biosensor containing the tandem C1 domains from protein kinase C-&#952; linked to GFP (C1-GFP) and RFP...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

The overall goal of this protocol is to describe the use of photoactivatable peptide MHC to induce polarized signaling in T lymphocytes. This method can help answer key questions in immune cell signal transduction such as how lymphocytes transduce rapid localized signals and then mount polarized cellular responses to those signals. The main advantage of this technique is that it offers spatial temporal control of T cell signaling.It does this by fixing the precise time and location of T cell receptor activation. Demonstrating the procedure will be Elisa Sanchez, a grad student from my laboratory. Begin by adding biotinylated poly-L-lysine, or bio-PLL, diluted one to 500 in distilled deionized water to eight-well chambered cover glass.Incubate for 30 minutes at room temperature. Following the incubation, wash with distilled deionized water and then dry for two hours at room temperature. Block bio-PLL coated surfaces with blocking buffer consisting of HEPES buffered saline with 2%BSA for 30 minutes at room temperature.Following the incubation, remove the blocking buffer from the bio-PLL coated surfaces without allowing the wells to dry. Then add streptavidin and incubate at four degrees Celsius. After an hour, wash the coated surfaces in HBS.Fill and invert the chamber slide wells four to five times, removing HBS from the wells. Do not allow the wells to dry. Add the specific biotinylated photoactivatable peptide major histocompatibility complex ligands and adhesion molecules for either CD4 positive T cells or CD8 positive cells to the bio-PLL coated surfaces.Protect from light and incubate for one hour at room temperature. After the incubation, wash as before and leave in HBS until ready to seed. Finally, add 200, 000 CD4 positive or CD8 positive T cells expressing the appropriate TCR into the correct wells and allow the cells to adhere at 37 degrees Celsius for 15 minutes.Once the cells have attached to and spread on the surface, they are ready for photoactivation and imaging. Use an inverted total internal reflection fluorescence microscope outfitted with a UV-compatible 150X objective lens for image acquisition. Monitor probes in both the green and red channels using 488 nanometer and 561 nanometer excitation lasers, respectively.After mounting the chamber slide containing the T cells, adjust the microscope settings to obtain TIRF or epifluorescence illumination, as necessary. In Live mode, select a field of cells that are expressing the fluorescent probes of interest. Establish micron scale regions for photoactivation beneath individual cells using software control.Then, start the acquisition. Typically, 80 time points are required with an interval of five seconds between each. This leaves time for sequential 488 nanometer and 563 nanometer exposures in the case of dual color experiments.Then, after acquiring 10 time points, photoactivate the selected regions by opening the digital diaphragm shutter for one to 1.5 seconds. After the time lapse is complete, select a new field of cells and repeat the process. Begin by determining the fluorescence intensity of a background region outside of the cell to be used for background correction.Draw a micron-sized square region outside of the cell and make a mask. To determine the fluorescence intensity, click on Analyze, Mask Statistics. Select Mean fluorescence intensity and export the values.Determine the fluorescence intensity within the photoactivated region for each time point. Select Mask to highlight the region that was photoactivated. To determine the fluorescence intensity, click on Analyze, Mask Statistics.Select Mean fluorescence intensity and Export the values. Obtain the X and Y coordinates of the center of the photoactivated region by selecting Mask to highlight the region that was photoactivated. Once the region of photoactivation is highlighted, select Analyze, Mask Statistics, Center of Area, and Export the values.Determine the X and Y coordinates of the fluorescent probe of interest. Select Manual Particle Tracking and click on the fluorescent probe of interest over time. Once all of the time points have been tracked, select Analyze, Mask Statistics, Center of Area, and Export the values.T cells were attached to chambered cover glass containing photoactivatable MCC-IEFK. C1-GFP, a probe for the lipid second messenger DAG, was imaged using TIRF microscopy and RFP-Tubulin in epifluorescence mode. Localized photoactivation of the surface beneath the T cell induces the accumulation of DAG in the irradiated zone.This is followed within seconds by the reorientation of the centrosome to the same region. C1-GFP accumulation can be quantified by calculating the normalized fluorescence intensity after background correction at the center of the photoactivated region over time. Centrosome reorientation in response to photoactivation can be quantified by calculating the distance between the centrosome and the center of the photoactivated region as a function of time.Once mastered, this technique can be done in less than a day if it is performed properly. We typically generate 15 to 20 time lapse movies for analysis in this time. This approach paved the way for researchers to explore the precise kinetics and polarization of activating signaling in T cells.It was also adapted to study inhibitory signaling in natural killer cells. As a result, we now have a better understanding of how communicative immune cell cell interactions are assembled and dissolved.

Research

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Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...