<meta charset="utf8"></head><body>用于检测 T 细胞中 Ca2+ 微结构域的高分辨率成像技术

<ol>
	<li>Wolf, I. M. 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=Frontrunners+of+T+cell+activation:+Initial,+localized+Ca2++signals+mediated+by+NAADP+and+the+type+1+ryanodine+receptor.">Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.</a> Sci Signal. 8 (398), ra102 (2015).</li><li>Taguchi, T., Mukai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+signalling+and+membrane+trafficking.">Innate immunity signalling and membrane trafficking.</a> Curr Opin Cell Biol. 59, 1-7 (2019).</li><li>Trebak, M., Kinet, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+in+T+cells.">Calcium signalling in T cells.</a> Nat Rev Immunol. 19 (3), 154-169 (2019).</li><li>Diercks, B. -. 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=ORAI1,+STIM1/2,+and+RYR1+shape+subsecond+Ca2++microdomains+upon+T+cell+activation.">ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation.</a> Sci Signal. 11, eaat0358 (2018).</li><li>Cheng, H., Lederer, W. J., Cannell, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sparks:+Elementary+events+underlying+excitation-contraction+coupling+in+heart+muscle.">Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle.</a> Science. 262 (5134), 740-744 (1993).</li><li>Feske, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+in+lymphocyte+activation+and+disease.">Calcium signalling in lymphocyte activation and disease.</a> Nat Rev Immunol. 7 (9), 690-702 (2007).</li><li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Kummerow, C., Junker, C., Kruse, K., Rieger, H., Quintana, A., Hoth, 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+immunological+synapse+controls+local+and+global+calcium+signals+in+T+lymphocytes.">The immunological synapse controls local and global calcium signals in T lymphocytes.</a> Immunol Rev. 231 (1), 132-147 (2009).</li><li>Brock, V. 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=P2X4+and+P2X7+are+essential+players+in+basal+T+cell+activity+and+Ca2++signaling+milliseconds+after+T+cell+activation.">P2X4 and P2X7 are essential players in basal T cell activity and Ca2+ signaling milliseconds after T cell activation.</a> Sci Adv. 8 (5), eabl9770 (2022).</li><li>Wei&#223;, 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=Adhesion+to+laminin-1+and+collagen+IV+induces+the+formation+of+Ca2++microdomains+that+sensitize+mouse+T+cells+for+activation.">Adhesion to laminin-1 and collagen IV induces the formation of Ca2+ microdomains that sensitize mouse T cells for activation.</a> Sci Signal. 16 (790), eabn9405 (2023).</li><li>Diercks, B. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+Ca2++microdomains+for+the+adaptive+immune+response.">The importance of Ca2+ microdomains for the adaptive immune response.</a> Biochim Biophys Acta Mol Cell Res. 1871 (5), 119710 (2024).</li><li>Woelk, L. -. 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=DARTS:+an+open-source+Python+pipeline+for+Ca2++microdomain+analysis+in+live+cell+imaging+data.">DARTS: an open-source Python pipeline for Ca2+ microdomain analysis in live cell imaging data.</a> Front Immunol. 14, 1299435 (2024).</li><li>Diercks, B. -. P., Werner, R., Schetelig, D., Wolf, I. M. A., Guse, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+calcium+imaging+method+for+local+calcium+signaling.">High-resolution calcium imaging method for local calcium signaling.</a> Methods Mol Biol. 1929, 27-39 (1929).</li><li>. Python Available from: <a target="_blank" href="https://www.python.org/downloads/">https://www.python.org/downloads/</a> (2024)</li><li>. Anaconda Available from: <a target="_blank" href="https://docs.anaconda.com/free/anaconda/install/index.html">https://docs.anaconda.com/free/anaconda/install/index.html</a> (2024)</li><li>. git Available from: <a target="_blank" href="https://git-scm.com/book/en/v2/Getting-Started-Installing-Git">https://git-scm.com/book/en/v2/Getting-Started-Installing-Git</a> (2024)</li><li>. DARTS Available from: <a target="_blank" href="https://github.com/IPMI-ICNS-UKE/DARTS">https://github.com/IPMI-ICNS-UKE/DARTS</a> (2024)</li><li>. DARTS GitHub Repository Available from: <a target="_blank" href="https://ipmi-icns-uke.github.io/DARTS/">https://ipmi-icns-uke.github.io/DARTS/</a> (2024)</li><li>. Oracle Available from: <a target="_blank" href="https://www.oracle.com/de/java/technologies/downloads/">https://www.oracle.com/de/java/technologies/downloads/</a> (2024)</li><li>. Time-Dependent Entropy Deconvolution Available from: <a target="_blank" href="https://ipmi-icns-uke.github.io/TDEntropyDeconvolution/General/2-usage.html#input-parameters">https://ipmi-icns-uke.github.io/TDEntropyDeconvolution/General/2-usage.html#input-parameters</a> (2024)</li><li>Gu, F., 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=Dual+NADPH+oxidases+DUOX1+and+DUOX2+synthesize+NAADP+and+are+necessary+for+Ca2++signaling+during+T+cell+activation.">Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.</a> Sci Signal. 14 (709), eabe3800 (2021).</li><li>Roggenkamp, H. 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=HN1L/JPT2:+A+signaling+protein+that+connects+NAADP+generation+to+Ca2++microdomain+formation.">HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation.</a> Sci Signal. 14 (675), eabd5647 (2021).</li><li>He&#223;ling, L. D., Troost-Kind, B., Wei&#223;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAADP-binding+proteins+-+Linking+NAADP+signaling+to+cancer+and+immunity.">NAADP-binding proteins - Linking NAADP signaling to cancer and immunity.</a> Biochim Biophy. Acta Mol Cell Res. 1870 (7), 119531 (2023).</li><li>Dynes, J. L., Amcheslavsky, A., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+targeted+single-channel+optical+recording+reveals+multiple+Orai1+gating+states+and+oscillations+in+calcium+influx.">Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx.</a> Proc Natl Acad Sci U S A. 113 (2), 440-445 (2016).</li></ol>

<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

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.

<meta charset="utf8"></head><body>作者聚焦：揭示治疗干预的适应性免疫反应的初始激活

原代 T 细胞中初始 Ca2+ 微结构域的成像

Our fundamental research, we want to understand the initial activation of the adaptive immune response. Calcium is one of the most widely used intracellular signal messengers. If we decipher the complex calcium activation, we may be able to intervene with a signaling machinery to treat pathological conditions such as autoimmune disease.Using this method, we were able to identify the key players in the NADP signal in cascade and T cells, such as the NADP producing enzyme and NADP binding protein. Moreover, we were able to show that adhesion dependent calcium micro domains contribute to initial calcium signals independent of T-cell receptor stimulation. Our method is the first capable of detecting small, fast and highly dynamic calcium micro domains in primary T-cells, and can be easily adapted to other cell types, or stimulations and inhibitions.Additionally, the combination of two chemical dyes allows the calibration of these calcium micro remains. We believe that the initial calcium micro remains shape the adaptive immune response by controlling the fate of individual T cells. Therefore, want to link the initial calcium response to downstream cycling events and adapt our imaging to life cell super resolution to resolve initial calcium nano domains.

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

Imaging Initial Ca2+ Microdomains in Primary T Cells

394 次观看.  University Medical Centre Hamburg-Eppendorf. 我们的基础研究，我们想了解适应性免疫反应的初始激活。钙是使用最广泛的细胞内信号信使之一。如果我们破译了复杂的钙激活，我们可能能够使用信号机制进行干预，以治疗自身免疫性疾病等病理状况。使用这种方法，我们能够识别级联细胞和 T 细胞中 NADP 信号的关键参与者，例如产生 NADP 的酶和 NADP 结合蛋白。此外，我们能够证明粘附依...