<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), 102 (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, 0358 (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), 9770 (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), 9405 (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 (2019).</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), 3800 (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), 5647 (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

<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

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

755 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...

视频: 作者聚焦：揭示治疗干预的适应性免疫反应的初始激活

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细胞受体在小鼠T细胞的逆转录病毒转导

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 ...

科研

免疫与感染

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.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 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 ...

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&amp;#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.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

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

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.

piggyBac转座子初级人类T细胞系统改造

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.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

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.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

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.

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

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.

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

实时监测细胞因子分化人原代T细胞中的线粒体呼吸

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi (T. cruzi), is an obligate intracellular parasite with a diverse biology that infects several mammalian species, including humans, causing cardiac and digestive pathologies. Reliable detection of T. cruzi in vivo infections has long been needed to understand Chagas disease&#39;s complex biology and accurately evaluate the outcome of treatment regimens. The current protocol demonstrates an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs. Light-sheet fluorescent microscopy allows for accurately visualizing and quantifying of actively proliferating and dormant T. cruzi parasites and immune effector cells in whole organs or tissues. Also, the CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled with 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues, and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage, and repair in Chagas disease.

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

The present protocol describes light-sheet fluorescent microscopy and automated software-assisted methods to visualize and precisely quantify proliferating and dormant Trypanosoma cruzi parasites and T cells in intact, cleared organs and tissues. These techniques provide a reliable way to evaluate treatment outcomes and offer new insights into parasite-host interactions.

完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像

Chagas disease is a neglected pathology that affects millions of people worldwide, mainly in Latin America. The Chagas disease agent, Trypanosoma cruzi ...