This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (project number 335447717; SFB1328, A02 to B-PD and RW; A14 to ET; project number 516286863 to B-PD). The authors thank the blood donors and the Department of Transfusion Medicine at the UKE for their cooperation.

All animal experiments were approved and performed in accordance with the animal welfare guidelines of the Institutional Animal Care and Use Committee at University Medical Centre Hamburg-Eppendorf.
1. Isolation of primary mouse T cells from lymph nodes and spleens
<ol>
	<li>Harvest the spleens/lymph nodes under sterile conditions according to the ethical guidelines and place them in a tube with ice-cold Clicks medium (10% Fetal Calf Serum (FCS), 2 mM L-Glutamine, Penicillin/Streptomycin (P/S) 100 U/mL, 50 nM &#946;-Mercaptoethanol). 
	NOTE: Wild type and KO mouse T cells are isolated in the same way.</li>
	<li>Cool down the centrifuge to 4 &#176;C.</li>
	<li>Place the spleen and lymph nodes in a 70 &#181;m cell strainer in a sterile Petri dish and add spleen isolation medium (RPMI medium + 7.5 % NCS + 1% P/S) to a total volume of 20 mL.</li>
	<li>Thoroughly but gently disrupt the spleen using a plastic mortar. Transfer the cells afterward to a 50 mL centrifuge tube. 
	NOTE: A total volume of 20 mL should now be in the 50 mL centrifuge tube. Spleen and lymph nodes should be completely disrupted. Work on ice from this point onwards. Keeping the cells on ice slows down their metabolism and prevents cell death, ensuring they remain viable and functionally intact. This helps to preserve their physiological state for a longer period of time, which is especially important for accurate downstream experiments.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and ensure resuspension of the resulting pellet in 2 mL of Dulbecco`s Phosphate Buffered Saline (DPBS) (without Ca2+ and Mg2+). Transfer to a new 12 mL centrifuge tube.</li>
</ol>
2. Negative selection of CD4+ T cells
NOTE: For the negative selection of CD4+ T cells, a T cell isolation kit containing a&#160;FcR blocker, biotinylated antibodies against non-CD4+ T cells, and streptavidin-coated magnetic particles are used.
<ol>
	<li>Add 20 &#181;L/mL of mouse FcR blocker from the T cell isolation kit to the cell suspension.</li>
	<li>Further, add 100 &#181;L of the mouse CD4+&#160;T cell isolation cocktails at a concentration of 50 &#181;L/mL, mix well, and incubate for 10 min at room temperature (RT).</li>
	<li>Vortex streptavidin-coated magnetic particles for 30 s, add 150 &#181;L to the cell suspension, mix well, and incubate for 2 min 30 s at RT.</li>
	<li>Add DPBS to the cell suspension, filling it to a total volume of 7 mL. Ensure to pipette up and down gently, then incubate in the magnet for 2 min 30 s at RT.</li>
	<li>Subsequently, gently collect the enriched T cells in the supernatant and transfer them to a new 12 mL centrifuge tube.</li>
	<li>Centrifuge cells at 300 x g at RT for 5 min.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend the isolated and enriched CD4+ T cells in 1 mL of DPBS, thoroughly pipetting up and down. Incubate the mixture for 2 min 30 s in the magnet at RT to ensure no bead contamination.</li>
	<li>Collect the supernatant, transfer it into a new centrifuge tube and count the enriched T cells.</li>
	<li>To count the cells, stain the cells with trypan blue and count using an automated cell counter. 
	NOTE: It is best to directly load the isolated CD4+ T cells and image them on the same day of isolation. However, primary murine T cells can be cultured without any stimulation overnight in an incubator at 37 &#176;C with 5 % CO2 in an isolation medium (RPMI 1640 + 10% FCS + P/S).</li>
</ol>
3. Loading of primary mouse CD4+ T cells
NOTE: To measure free cytosolic Ca2+ concentrations, fluorescent and membrane-permeable dyes are employed in the Ca2+ imaging experiments. A combination of Fluo-4- acetoxymethyl ester (AM) and the ratiometric dye Fura Red-AM serves as an indicator for rapid detection of local Ca2+ signals. Ensure to work in the dark while using fluorescent dyes.
<ol>
	<li>Centrifuge 2-5 x 106 cells for 5 min, 300 x g, discard the supernatant and resuspend in 480 &#181;L of T cell medium (RPMI 1640, 10% FCS) with 10 &#181;M Fluo-4-AM (Stock: [1 mM]), and 20 &#181;M Fura Red-AM (Stock: [4 mM]).</li>
	<li>Incubate initially for 20 min at RT under the bench in the dark, covering the falcon with aluminum foil. 
	NOTE: Do not exceed the first incubation time, as the Ca2+ indicators are diluted in DMSO, which could be harmful to the cells at a longer exposure time in the low total volume. Keep the cells shielded from light after loading due to the light sensitivity of the dyes.</li>
	<li>At the end of the initial 20-min incubation, add 2 mL of T cell medium to the cells and continue the incubation for an additional 30 min in the dark at RT.</li>
	<li>Centrifuge the cell suspension at 300 x g for 5 min at RT.</li>
	<li>Discard the supernatant and cleanse the pellet by washing it with 2 mL of Ca2+ measuring buffer (140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM HEPES, 1 mM NaH2PO4, 5 mM glucose, pH 7.4) followed by centrifugation at 300 x g for 5 min at RT.</li>
	<li>Finally, discard the supernatant and resuspend it with the Ca2+ measuring buffer. Adjust the cell number to ~100,000 cells per 10 &#181;L. 
	NOTE: A volume of 10 &#181;L is used for one measurement. Avoid overdiluting the cells, as one might need to further adjust the cell count under the microscope to reach an optimum for the measurement conditions.</li>
	<li>Allow the cells to incubate for ~20 min to ensure complete cell infiltration and de-esterification of the dyes. Store cells on ice and in the dark until measurement. 
	NOTE: Loaded primary murine T cells can be used up to ~4 h after loading.</li>
</ol>
4. Local Ca2+ imaging
<ol>
	<li>Slide preparation
	<ol>
		<li>Coat microscope coverslips (24 mm x 46 mm) with bovine serum albumin (BSA, 5 mg/mL) and poly-L-lysine (PLL, 0.1 mg/mL) (Figure 1A). Allow BSA to sit for ~20 min before applying PLL. 
		NOTE: Coating facilitates cell adhesion. For proper cell adhesion, accurately spread both BSA and PLL until no more streaks are visible.</li>
		<li>To create reaction chambers, glue reusable rubber O-rings to the slides using a silicone paste (Figure 1B,C). 
		NOTE: Other slides/chambers or plates such as 35 mm slides, reusable metal chambers, or 8-, 24- or 48-well plates can be used for image acquisition.</li>
	</ol>
	</li>
	<li>Coating of Protein G magnetic beads (10 &#181;m in diameter) with antibodies
	<ol>
		<li>Gently mix protein magnetic bead suspension and transfer 12.5 &#181;L to a new tube.</li>
		<li>Place the tube on a magnetic stand to remove the storage buffer and let the beads migrate to the magnet. Gently remove the storage buffer by pipetting it on the opposite side of the magnet.</li>
		<li>To remove any remaining storage buffer, wash the beads by adding 500 &#181;L of PBS-T (PBS, 0.1% Tween) and vortexing for 10 s. Place the tube in the magnetic stand again and remove the buffer.</li>
		<li>To coat the beads with antibodies, resuspend them in 7.5 &#181;L of PBS-T and add 5 &#181;L of anti-CD3 (0.5 mg/mL) and anti-CD28 (0.5 mg/mL), respectively. Incubate for 30-60 min with continuous mixing at RT.</li>
		<li>Wash the coated beads three times with 500 &#181;L of PBS-T, followed by washing once with 500 &#181;L of Ca2+&#160;measuring buffer using the magnetic stand.</li>
		<li>Resuspend beads in 200-400 &#181;L of Ca2+&#160;measuring buffer. 
		NOTE: Check bead density during measurements, further dilute if necessary.</li>
	</ol>
	</li>
	<li>Local Ca2+ imaging microscopy 
	NOTE: Imaging is performed using a brightfield light microscope with a 100-fold magnification, equipped with a xenon arc lamp as a light source. Frames are captured in 14-bit mode with two-fold binning using an electron-multiplying charge-coupled camera. To record and split the emission wavelengths of both dyes, a dual-view module is employed, featuring the following filters in nm (ex, 480/40; bs, 495; em1, 542/50; em2, 650/57). The imaging setup includes an acquisition hub and imaging software for image acquisition.
	<ol>
		<li>Place 10 &#181;L of the loaded cells on the prepared slide and let them attach to the slide for 3-5 min.</li>
		<li>Gently add 80 &#181;L of Ca2+ measuring buffer to the slide.</li>
		<li>Select the 100x oil immersion lens and apply a small drop of immersion oil. Place the slide on the microscope table.</li>
		<li>Adjust the focus in the brightfield modus, carefully select a field of view with up to 10 cells that are not in contact with each other, and acquire an image. 
		NOTE: Overlapping and touching cells will be hard to analyze later.
		<ol>
			<li>Turn on the lamp, check the fluorescence and the loading of the T cells, and compare cells in both channels to verify that they are not preactivated.</li>
			<li>Ensure to take pictures of the field of view for both brightfield and fluorescence channels before and after the measurement to check for cell movement and loading.</li>
		</ol>
		</li>
		<li>Start measurement and capture the basal activity for 1 min with an acquisition rate of 1 frame per 5 s.</li>
		<li>Add 10 &#181;L of compound/stimulant (bead or stimulator/inhibitor) after 1 min and measure for a total of 3 min using 40 frames per s or the maximum frame rate possible. 
		NOTE: The addition of beads is the crucial step; ensure that the beads are added close to the excitation light on the slide while not moving the slide. Representative examples of Ca2+ microdomains in primary CD4+ T cells upon bead contact from WT and P2x4-/- and P2x7-/- mice are shown in Figure 2.</li>
	</ol>
	</li>
</ol>
5. Postprocessing/ data analysis
NOTE: For image processing and data analysis, the Python-based open-source pipeline DARTS is used. It has been developed by Woelk et al.12 based on work by Diercks et al.13.
<ol>
	<li>Installation of the DARTS pipeline
	<ol>
		<li>Install Python 3.10.014, anaconda15, and git16, and clone the GitHub repository using the terminal command git clone: https://github.com/IPMI-ICNS-UKE/DARTS.git</li>
		<li>Create a conda environment with conda create --name DARTS and install all necessary Python packages with pip install &#60;package&#62;. 
		NOTE: Follow the installation instructions in the DARTS GitHub repository17,18 for more detailed information.</li>
		<li>Before using Bioformats, ensure that a Java Runtime Environment is correctly installed19.</li>
	</ol>
	</li>
	<li>Usage of the DARTS pipeline 
	NOTE: Once installed, DARTS can be started from the terminal window.
	<ol>
		<li>Navigate to the local copy of the DARTS repository (cd path/to/DARTS) that contains the main.py file. Make sure that the conda environment is activated (conda activate DARTS).</li>
		<li>Execute DARTS by typing python main.py in the terminal window and pressing Enter.</li>
		<li>In the graphical user interface (GUI), specify the source and results directory as well as the image format in the upper left section (see Figure 3).</li>
		<li>Define the properties of measurement, such as the scale (pixels per micrometer), frame rate, cell type, etc. The processing pipeline can be assembled based on the specific image data and research question. 
		NOTE: Read more about the deconvolution parameters, for instance, in the corresponding documentation20.</li>
		<li>Save the settings on the computer before clicking on Start. A more detailed description of the GUI can be found in the DARTS documentation.</li>
	</ol>
	</li>
	<li>Ca2+ microdomain imaging and bead contacts 
	NOTE: For local imaging and stimulation with beads and subsequent image analysis, the cell-specific bead contacts need to be defined by the user to identify the starting point (t = 0) of the measurement period of interest (see Figure 4). A bead contact is defined as the contact site of a (stimulatory) bead with a cell of interest. It consists of the specification of (1) the bead contact time, e.g., frame 300, and (2) the bead contact position relative to the center of the cell, e.g., 1 o&#39;clock or 12 o&#39;clock. With the current DARTS version, the user needs to manually define and enter the information for each bead contact. Use the following procedure to define bead contacts:
	<ol>
		<li>Use the slider to pinpoint the time of contact between a bead and a cell of interest.</li>
		<li>In the options menu on the right-hand side, select bead contact: x, y, t.</li>
		<li>Select the spot on the left side of the image where the cell and bead make contact.</li>
		<li>Select Choose cell by clicking a point inside. Click on the cell stimulated by this bead, preferably in the center.</li>
		<li>Click on ADD bead contact.</li>
		<li>Repeat steps 5.3.1- 5.3.5 for each additional bead contact in this file. Once all bead contacts are defined, click on the Continue button.</li>
		<li>Proceed with additional files. After the last file is reached, the script will automatically initiate analysis of all files.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Find the processed ratio images, microdomain data for each cell over time (incl. localization, amplitude, size), source data for the dartboard projection and further resources in the results folder.</li>
		<li>To create dartboards, navigate to the folder /src/analysis/ inside the DARTS folder (pattern: cd path/to/DARTS/src/analysis) containing the script DartboardPlotGUI.py. Then, type python DartboardPlotGUI.py and press enter.</li>
		<li>Ensure that the necessary information is provided and that the spreadsheet files from the results folder are accurately selected as the source files for dartboard generation.</li>
	</ol>
	</li>
</ol>

High-Resolution Imaging Technique for Detecting Ca2+ Microdomains in T Cells

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The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be considered a potential conflict of interest.

We described an extensive protocol for high-resolution live cell imaging of local Ca2+ microdomains in primary murine and human T cells triggered by TCR/CD3 stimulation through antibody-coated beads. Moreover, we implemented a user-friendly and open-source Python-based algorithm to identify and analyze local Ca2+ signals. Notably, the protocol is not limited to the detection of Ca2+ microdomains in the context of TCR/CD3 stimulation but is adaptable to other (immune) cell types such as NK cell lines (KHYG-1)12 or TCR-independent Ca2+ microdomains10,11.
A critical step within the protocol is the size and number of the stimulating beads. To mimic an immune-synapse, the beads should be similar in size to the cells. Hence, for primary murine and human T cells as well as cell lines (Jurkat and KHYG1), we use magnetic beads with a diameter of 10 &#181;m. Furthermore, each cell should be just stimulated by one single bead. Therefore, the number of beads added to each slide should be sufficient on the one hand, but if there are too many beads in the field of view, the background increases, and it is not possible to detect a single activation time point and contact side.
The protocol utilizes the fluorescent Ca2+ dyes Fluo-4 AM and FuraRed AM in a ratiometric manner, therefore allowing for calibration of the data13. Additionally, the protocol could be adapted to other Ca2+ indicator pairs, but caution must be taken in the selection process in terms of Ca2+ binding kinetics, subcellular distribution, and photobleaching1. Moreover, loading conditions must be developed and optimized for each individual cell type, but the concentrations indicated here are a good starting point. To visualize Ca2+ microdomains, the Kd of the Ca2+ dyes should be in the range of 300-1200 nM&#160;and the acquisition time per frame should be &#8804;60 ms. If the fluorescence intensity is too low, the filter set has to be checked, but it is also possible to load a double amount of Ca2+ dye into T cells. However, the Ca2+ dye could mislocate to other organelles or sequester to vesicles, but it might also act as a Ca2+ buffer and affect the Ca2+ responses.
One limitation of the analysis algorithm is that a spherical shape of the cell is assumed; hence, cell types with different morphologies might need adaption of the analysis toolbox. The algorithm has been used to analyze local Ca2+ microdomains in primary murine T cells, as well as Jurkat T cells and an NK cell line (KHYG-1)12&#160;and was successful in the analysis of Ca2+ microdomains for a murine neuronal cell line (N2a, unpublished data). In principle, the protocol and analysis toolbox could be used to analyze non-spherical cell types such as HEK293 or HeLa cells, but for these cell types, the dartboard projection cannot be adapted because it is based&#160;on a round structure and shape normalization of the cells. In addition, the protocol to detect localized initial Ca2+ microdomains upon bead stimulation can be adapted to analyze local Ca2+ signals derived from other stimuli, such as soluble activating or inhibiting compounds, as well as adhesion-dependent and TCR/CD3 independent Ca2+ microdomains10,11. Of note, it is easier to define a single bead contact in terms of time and location than to determine the starting point of activation after soluble compounds.
A general limitation for the detection of Ca2+ microdomain formation lies in the required high temporal-spatial resolution and necessary high signal-to-noise ratio (SNR). Currently, the resolution derived from our setup reaches a calculated spatial resolution of ~0.368 &#181;m and temporal resolution of ~40 frames per second (fps)1. Recent advances in camera and detector development, as well as improvement of fluorescent dyes, might lead to the possibility of reaching optical single channel recordings as they have been described for ORAI-GECI (genetically expressed Ca2+ indicators) constructs24 for live cell imaging using Ca2+ indicators with higher temporal and spatial resolution in the future.
Taken together, the protocol and analysis tool for high-resolution Ca2+ microdomain imaging described here can be used not only to analyze initial local Ca2+ signals in T cells but can also be adapted to other cell types to decipher the significance of local Ca2+ signaling in these.

The presented fluorescence microscopy technique enables the visualization of local, temporal initial calcium (Ca2+) signals in primary mouse T cells, termed Ca2+ microdomains. Ca2+ microdomains represent highly dynamic and short-lived Ca2+ signaling events, posing challenges for effective live cell imaging and analysis1.
T cells are challenging for live cell imaging due to the relative differences in central and peripheral fluorescence intensity, which can be attributed to their spherical shape and their small diameter of ~6-8 &#181;m. Upon stimulation and immune synapse formation, T cells undergo morphological changes, further complicating the imaging of T cells1. Therefore, employing a ratiometric analysis becomes imperative, achieved by either recording two images representing different properties of a Ca2+ dye or utilizing a combination of two Ca2+ dyes. The demanding characteristics of Ca2+ microdomains include their rapid, temporally, and spatially limited nature. To capture this, the Ca2+ dyes used must possess both a high basal brightness and a high signal-to-noise ratio (SNR) to obtain the highest temporal and spatial resolution possible. Optimal results have been achieved using a combination of the dual-wavelength dye Fura Red and the single-wavelength dye Fluo-4. Co-loading cells with Fluo-4 and Fura Red mitigate the challenges posed by strong photobleaching of dual-emission wavelength dyes and the temporal delay associated with dual-excitation dyes, ensuring suitability for fast image acquisition. This approach further facilitates the visualization of changes in shape and subtle movements. Special demands are also placed on the imaging system in terms of spatial resolution to enable the visualization of Ca2+ signals originating from the opening of small clusters of channels or even single channels1.
Ca2+ signaling plays a pivotal role in activating immune functions within T cells, including synapse formation and cytokine production and release2,3. The specific fate of the cell is regulated through the differently pronounced and locally distributed Ca2+ signals, the Ca2+ microdomains3. Notably, those local Ca2+ signals precede a widespread rise in intracellular Ca2+ levels in T cells&#160;and the formation of Ca2+ microdomains depends on both Ca2+ entry and release1,4,5. Upon T cell receptor (TCR)/CD3 stimulation, the formation of Ca2+ releasing second messengers, like nicotinic acid adenine dinucleotide phosphate (NAADP), D-myo-inositol 1,4,5-trisphosphate (IP3) and cyclic ADP ribose (cADPR) is triggered, leading to rising intracellular Ca2+ levels up to 1 &#181;M6,7. Early Ca2+ signaling events are linked to the release of Ca2+ from intracellular Ca2+ stores such as the endoplasmic reticulum (ER), with channels such as the ryanodine receptor 1 (RyR1) and the IP3 receptor (IP3R) being predominantly responsible for this signaling. This subsequently triggers extracellular Ca2+ influx and results in a global Ca2+ signal via store-operated Ca2+ entry (SOCE)8. In addition, there are other channels involved in Ca2+ signaling during T cell activation9, e.g., P2X4 and P2X7 channels ensure adenosine triphosphate (ATP)-dependent cation influx, contributing to the rise in intracellular Ca2+. Remarkably, initial adhesion-dependent Ca2+ microdomains (ADCMs) are already formed&#160;before TCR stimulation but with lower Ca2+ amplitudes and frequencies. These initial TCR-independent Ca2+ signals most likely serve the migration of the T cell to the site of inflammation and prime the T cells for restimulation at the site of infection10,11.
By developing the described method for local Ca2+ imaging, we have gained an additional tool for exploring the origin and significance of early Ca2+ signals in T-cell activation. This method enables the user to detect smaller, short-lived, and more rapidly occurring Ca2+ signals than&#160;previously possible. Moreover, Deconvolution, Analysis, Registration, Tracking, and Shape normalization (DARTS), the Python-based analysis pipeline, enables sharing the analysis tools with a broader audience12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >&#945;-CD3</td>
			<td >BD Pharmingen</td>
			<td >553058</td>
			<td ></td>
		</tr>
		<tr >
			<td >&#945;-CD28</td>
			<td >BD Pharmingen</td>
			<td >553295</td>
			<td></td>
		</tr>
		<tr >
			<td >Anaconda</td>
			<td>Anaconda</td>
			<td>https://docs.anaconda.com/free/anaconda/install/index.html&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bovine serum albumin</td>
			<td >Sigma-Aldrich</td>
			<td >A2058</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell strainer</td>
			<td>Biofil</td>
			<td>CSS-013-070</td>
			<td>70 &#181;m diameter</td>
		</tr>
		<tr >
			<td >Countess Automated Cell Counter</td>
			<td >Invitrogen</td>
			<td >A49865</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover slips</td>
			<td>Paul Marienfeld GmbH</td>
			<td>101202</td>
			<td></td>
		</tr>
		<tr >
			<td >DARTS</td>
			<td>GitHub</td>
			<td>https://github.com/IPMI-ICNS-UKE/DARTS&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dulbecco&#8217;s Phosphate buffered saline</td>
			<td>Gibco, Thermo Fisher</td>
			<td >14190144</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep CD4+ T cell Isolation Kit</td>
			<td >Stemcell</td>
			<td>19852</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep Magnetic Multistand</td>
			<td >Stemcell</td>
			<td>18010</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluo 4-AM</td>
			<td>Invitrogen</td>
			<td >F14201</td>
			<td></td>
		</tr>
		<tr >
			<td >Fura Red-AM</td>
			<td>Invitrogen</td>
			<td >F3020</td>
			<td></td>
		</tr>
		<tr >
			<td >Gibco RPMI 1640 medium</td>
			<td>Gibco, Thermo Fisher</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr >
			<td >Git</td>
			<td>GitHub</td>
			<td>https://git-scm.com/book/en/v2/Getting-Started-Installing-Git</td>
			<td ></td>
		</tr>
		<tr >
			<td >immersion oil</td>
			<td >Leica</td>
			<td >11513859</td>
			<td></td>
		</tr>
		<tr >
			<td >Newborn calf serum</td>
			<td>Sigma-Aldrich</td>
			<td >N4637</td>
			<td></td>
		</tr>
		<tr >
			<td >Oracle</td>
			<td>Oracle</td>
			<td>https://www.oracle.com/de/java/technologies/downloads/&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin/Streptamycin</td>
			<td>Gibco, Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr >
			<td >Poly-L-lysine</td>
			<td >Sigma-Aldrich</td>
			<td >P4707</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein G magnetic beads</td>
			<td >Merck</td>
			<td >LSKMAGG02</td>
			<td>10 &#181;M diameter</td>
		</tr>
		<tr >
			<td >Python</td>
			<td>Python</td>
			<td>https://www.python.org/downloads/&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >Silicon grease (basylone)</td>
			<td >Bayer</td>
			<td >291-1220</td>
			<td></td>
		</tr>
		<tr >
			<td >Time-Dependent Entropy Deconvolution</td>
			<td>GitHub</td>
			<td>https://ipmi-icns-uke.github.io/TDEntropyDeconvolution/General/2-usage.html#input-parameters</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tween</td>
			<td >q-biogene</td>
			<td >TWEEN201</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging initial ca2+ microdomains in primary t cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

In this protocol, we outlined an updated method to image and analyze initial Ca2+ microdomains in primary mouse T cells based on previous work by our group1,13. This approach was instrumental in unraveling the involvement of CRAC channels such as ORAI1, STIM1, and STIM2, as well as intracellular Ca2+ release channels like RyR1 in early Ca2+ signaling events4.
To do so, we investigated spontaneous Ca2+ microdomain formation by imaging non-stimulated primary mouse Orai1-/-, Stim1-/-, Stim2-/-, and Ryr1-/- and compared them to WT primary mouse T cells. The analysis of Ca2+ microdomain formation encompassed signal onset velocity, Ca2+ amplitude, and number of signals per confocal plane. Notably, except for Stim2-/- T cells, all KO T cells displayed a significant decrease in local Ca2+ signals and a reduced basal free cytosolic Ca2+ concentration compared to WT cells. This led us to conclude that the formation of Ca2+ microdomains is intricately linked to the interaction of ORAI1, STIM1, and RyR14. In addition, we successfully identified and characterized spontaneous Ca2+ microdomains at the plasma membrane. These Ca2+ microdomains were characterized by a Ca2+ amplitude of 290 nm &#177; 12 nm. Utilizing a color-coded approach for Ca2+ signals allowed for the visualization of Ca2+ microdomains across the cell. Results further highlighted the rapid onset of Ca2+ microdomains, visible within milliseconds, and the capability of this method to detect Ca2+ signals with a longevity of a few milliseconds4. These spontaneous Ca2+ microdomains were later identified as adhesion-dependent Ca2+ microdomains (ADCM), dependent not only on SOCE but also acting via the FAK/PLC-&#947;/IP3 signaling cascade10 and involvement of P2X49. Furthermore, this technique was fundamental in confirming dual oxidases 1 and 2 (DUOX1/2) as NAADP-producing enzymes21 and HN1L/JPT222 as one of the newly discovered NAADP-binding proteins23.
Figure 2&#160;shows representative examples of Ca2+ microdomains in primary CD4+ T cells upon bead contact from WT as well as P2x4-/-&#160;and P2x7-/-&#160;mice. Cells were loaded with the Ca2+ dyes Fluo-4 AM and Fura Red AM and imaged at an acquisition rate of 25 ms (40 frames/s). To mimic T cell synapse formation, cells were stimulated with anti-CD3/anti-CD28 coated beads. Initial Ca2+ microdomain formation was analyzed 1 s prior and up to 15 s after bead contact using the DARTS pipeline. Upon bead contact, the WT cell showed rapid formation of Ca2+ microdomains in the first second after stimulation at the bead contact site (Figure 2A). These Ca2+ microdomains further expanded throughout the cell in the following 15 s after bead contact. Opposed to the WT cell, the P2x4-/-&#160;and P2x7-/-&#160;cells (Figure 2B) showed decreased Ca2+ microdomain formation upon bead stimulation, as well for the P2x4-/- a lower basal level before bead contact. These representative findings are in line with the previously published results by Brock et al.9, indicating Ca2+ microdomain formation in WT T cells directly after bead contact over 15 s and lower signals per frame in P2x4-/-&#160;and P2x7-/- cells. Moreover, the amplitude in P2x4-/- cells was significantly reduced, further establishing the role of purinergic signaling in adhesion-dependent Ca2+ microdomains.
Furthermore, this method can also be used to visualize initial Ca2+ microdomains in primary human CD4+ T cells (Figure 3). In line with primary murine T cells, initial Ca2+ microdomains are evoked at the bead contact site. However, the overall Ca2+ response appears to occur on a different timescale compared to murine CD4+ T cells.
The analysis of local Ca2+ signals in a manual manner is not feasible as it is quite laborious and subjective to the individual investigator. Therefore, we previously developed an algorithm in MATLAB Simulink using its image processing and optimization toolboxes for postprocessing13 for analyzing local Ca2+ microdomains.
Recently, we developed a new, open-source, postprocessing pipeline called DARTS for Ca2+ microdomain analysis in high-resolution live cell imaging using the software platform Python12. Here, different deconvolution algorithms can be selected, depending on the user&#39;s preference, a cell shape normalization performed to compensate for morphological cell shape changes, and microscope and measurement specific parameters defined (e.g., scale, frame rate, time measured) (Figure 4).
After selecting parameters for Ca2+ microdomain analysis, a second pop-up window is opened for each individual measurement to define the bead contact (Figure 5). To define the bead contact, the user can manually scroll through the tiff file using the slider and select the bead contact frame individually. Bead contact is selected by clicking at the bead contact site (Figure 5, bead and bead contact indicated by yellow ring and arrow) as well as cell selection. This step must be repeated for each cell of interest. Finally, the automated image postprocessing is applied&#160;and the results data is summarized and saved in a spreadsheet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67075/67075fig01.jpg" /> 
Figure 1: Workflow of slide preparation for imaging. (A) Add and spread both BSA and PLL on the slide using a second glass cover slip. (B,C) To build a chamber, glue the rubber o-rings using silicon grease onto the slide. Ensure that the whole ring is covered with a thin layer of grease to have proper isolation of the chamber. <a href="https://www.jove.com/files/ftp_upload/67075/67075fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67075/67075fig02.jpg" /> 
Figure 2: Representative cells of T cell receptor-dependent Ca2+ microdomains in a primary murine wild type (WT) (A), P2x4-/- or P2x7-/- (B) CD4+&#160;T cell. CD4+ T cells were negatively isolated and loaded with Fluo-4 AM and Fura Red, as described above. T cells were analyzed using the DARTS pipeline, resulting in comparable cell images to previously published results9. (A) WT primary T cell 1 s before stimulation with anti-CD3/anti-CD28 coated beads and up to 15 s after stimulation (scale bar 5 &#181;m), as well as 3D surface plot of a zoom-in from 0 s to 0.65 s in the bead contact region (scale bar 1 &#181;m). (B) Upper lane: representative P2x4-/- primary T cell 1 s before and up to 15 s after bead stimulation. Lower lane: representative P2x7-/- primary T cell 1 s before and up to 15 s after bead stimulation. <a href="https://www.jove.com/files/ftp_upload/67075/67075fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67075/67075fig03.jpg" /> 
Figure 3: Ca2+ microdomains in a representative primary human T cell after TCR stimulation. Primary human&#160;CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by fluorescence-activated cell sorting (FACS) from buffy coats and loaded with Fluo-4 AM and Fura Red, as described above. The figure shows a primary human&#160;T cell 1 s before stimulation with anti-CD3 coated beads and up to 15 s after stimulation (scale bar 5 &#181;m), as well as 3D surface plot of a zoom-in from 6.25 s to 7.5 s in the bead contact region (scale bar 1 &#181;m). <a href="https://www.jove.com/files/ftp_upload/67075/67075fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67075/67075fig04.jpg" /> 
Figure 4: The DARTS graphical user interface (GUI). The GUI is divided into four areas. In the Input/Output&#160;area, you need to provide information about the raw data, including the source directory and image configuration (either two channels per file or separate channels), as well as the results directory. In the Properties of Measurement&#160;area, the experiment needs to be described with all its relevant information, such as scale (microns per pixel), frame rate, and measurement interval relative to the later determined starting point. Next, a processing pipeline consisting of postprocessing steps, shape normalization, and the actual analysis (microdomain detection and dartboard data accumulation) can be assembled. Finally, the settings can be saved to or loaded from the computer. Once the analysis has been set up, click on Start to proceed. To read more about the setup, visit https://ipmi-icns-uke.github.io/DARTS/General/Usage.html.&#160;<a href="https://www.jove.com/files/ftp_upload/67075/67075fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67075/67075fig05.jpg" /> 
Figure 5: Manual definition of bead contacts. If beads are added to the cells during the experiment, the initial bead contact time with a cell of interest and the contact location have to be manually defined. This is done by scrolling through the frames with the slider and finding a position (x,y) at a time point t. To automatically fill the bead contact information field, the user clicks on the left half of the microscope image at the bead contact position. Next, to associate a cell with the bead contact, the user clicks on a position within the cell that has a bead contact. The information has to be confirmed by selecting ADD bead. <a href="https://www.jove.com/files/ftp_upload/67075/67075fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

imaging-initial-ca2-microdomains-in-primary-t-cells

Research

JoVE Journal

Immunology and Infection

790 Views.  University Medical Centre Hamburg-Eppendorf. Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Video: Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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

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

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

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

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

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

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

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+ ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.