Name: visualization of the immunological synapse by dual color time-gated stimulated emission depletion (sted) nanoscopy
Uploaded: 2014-03-24T20:00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy at JoVE.com

Vi takker Geoff Daniels for teknisk assistance. Dette arbejde blev finansieret af R01 AI067946 til JSO

1.. Frakke Dækglas med antistof <ol><li> Forvarm (ved 37 ° C) i 30 ml RPMI 10% FCS medier og 1 ml BD Cytofix / Cytoperm. </li><li> Der fremstilles en opløsning af 5 ug / ml oprenset antistof i phosphatbufret saltvand (PBS). Anbefales til aktivering af NK92 cellelinie, brug af anti-CD18 og anti-NKp30. <ol><li> Mark en cirka skilling mellemstore cirkel for hver tilstand på en # 1.5 dækglas ved hjælp af en PAP pen. For en dobbelt farve eksperiment, bør der være fire betingelser: uplettet, dobbelt farves, og to enkeltsenge farvede forhold. Doser 200 pi antistofopløsning i hver region, og der inkuberes ved 37 ° C i 30 minutter. </li><li> Vask dækglas ved forsigtigt at nedsænke hver en i 50 ml PBS i et 50 ml konisk rør ved stuetemperatur. Vask skal ske umiddelbart før tilsætningen af ​​bør tages celler og forsigtighed for at undgå antistof tørring på dækglasset. </li></ol></li></ol> 2. Aktiver NK-celler på Coverpodekviste; Fix og permeabiliserer <ol><li> Isoler 5 x 10 5 NK92 celler pr tilstand. Centrifuge og dekantere supernatant. Vask en gang med 10 ml forvarmet medier fra trin 1.1. Centrifuge og dekantere supernatant. <ol><li> Resuspender cellerne i forvarmede medier fra trin 1.1 i en koncentration på 2,5 x 10 6 / ml. </li><li> Forsigtigt dekanteres 200 ul til midten af ​​regionen skabt i afsnit 1.2.1. Inkuberes ved 37 ° C i 20 min ved 5% CO 2. (Bemærk: denne gang kan udvides eller mindskes, afhængigt af den biologiske funktion af interesse for NK-celle granula polarisering, 20 min er tilstrækkelig.). </li></ol></li><li> Efter inkubation af celler vaskes forsigtigt dækglas ved at dyppe hver i 50 ml stuetemperatur PBS i et 50 ml konisk rør. </li><li> Tilsæt 1 ul Triton X-100 til 1 ml forvarmet Fix / Perm opløsningen fra trin 1.1 og vortex grundigt. Lave og permeabilisere ved tilsætning af 200 pi Fix / Perm buffer (trin 2.3) til celler. Inkuber i 10 mi i mørke ved stuetemperatur. </li></ol> 3. Stain Cells <ol><li> Forbered farvning buffer: phosphatbufret saltvand (PBS), 1% BSA, 0,1% Saponin. <ol><li> Forbered opløsning af primært antistof i 200 pi farvning buffer (se trin 3.1). (Bemærk: antistof bør titreres før brug). Undgå brug af primært antistof, der er rejst i de samme arter, der anvendes til at belægge dækglasset (trin 1.2). Også undgå Strepatividin-biotin-bindinger til STED billeddannelse. </li><li> Efter punkt 2.3.1, forsigtigt vaske dækglas i 50 ml farvning buffer. Dup kanter PAP-pen region med vatpind til at fjerne overskydende buffer. Påfør antistof løsning skabt i afsnit 3.1.1. Inkuber 30 minutter i mørke ved stuetemperatur. (Anbefalet: inkuberes dækglas i dias kasse med en fugtig papirserviet for at bevare fugtighed). </li></ol></li><li> Forbered løsning af sekundært antistof i 200 pi farvningpuffer. Anbefalede fluorophores er Alexa Fluor 488, Pacific Orange, og V500. Generelt er en fortynding på 1:200 er egnet til STED billeddannelse. <ol><li> Vask forsigtigt dækglas i 50 ml farvning buffer. Dup kanter PAP-pen region med vatpind til at fjerne overskydende buffer. Anvend sekundært antistof opløsning. Inkuber 30 minutter i mørke ved stuetemperatur. </li></ol></li><li> Gentag vask og farvning for yderligere proteiner af interesse. Hvis påvisning af F-actin med phalloidin, kan dette indgå med sekundært antistof, generelt ved en 1:200 fortynding. </li></ol> 4.. Mount Dækglas på dias <ol><li> Forbered montage medium. Bemærk: Forlæng eller forlænge Gold er at foretrække. Vectashield skal undgås, da det ikke er foreneligt med STED. 2,2-thiodioethanol skal undgås, hvis phalloidin anvendes. Mowiol er acceptabel. </li><li> Placer cirka 10-20 ul montering medium på et dias. Invert dækglas (celle nedad) og montere dækglas forsigtigt, idet manundgå indføring af luftbobler. Inkuber slides i 24 timer (dækglasset op) før billeddannelse. </li><li> Seal kanter dækglas med neglelak. </li></ol> 5.. Forsøgsopstilling <ol><li> Igangsætte krævede lasere og software. Igangsætte STED udtømning laser ved 100% effekt. Juster STED laser, som i tilfælde af kommercielle systemer er ofte en automatiseret procedure. </li><li> Fokus prøven, begyndende med en enkelt farvede kontrol på mikroskopet med okularer. </li></ol> 6.. Optimering af indstillinger <ol><li> Scan den første kanal og optimere laser magt, exciteringsstrålen position og detektor rækkevidde. Hvis det er muligt, undgå en gevinst på&gt; 100. Fange billedet i konfokal at optimere indstillingerne. Line og / eller ramme gennemsnitsberegning vil øge opløsningen. Check for pixel mætning. Bemærk: Nogle mætning er acceptabelt i konfokal som anvendelsen af ​​STED vil reducere intensiteten af ​​emission. For STED vil en optimal pixelstørrelse være under 30 nm, hvordanstadigt bedre opløsning opnås med mindre pixel størrelser. Størrelsen af ​​området af interesse, der afbildes, vil diktere den nedre grænse for pixelstørrelse. Mindre pixel størrelser kan øge fotoblegning. <ol><li> Påfør STED udtømning stråle og fange billedet, startende med 50% udtynding laser magt. Hvis en forbedring i opløsningen er set, kan anvendes mere udtømning lasereffekt. På dette tidspunkt, kan det være nødvendigt at justere excitation laser magt, line gennemsnit, og / eller gevinst. </li><li> Anvend tid gating at reducere baggrund (mindst 0,3 nanosekunder). Juster indstillingerne, indtil en forbedring i opløsning i konfokal kan ses. Opløsning kan tilnærmes ved at estimere fuld bredde halv maksimum (FWHM). Dette repræsenterer afstanden ved halv maksimal intensitet af en Gauss top skabt ved at trække en linie nu over strukturen af ​​interesse, og er en almindeligt anvendt metode til at beregne opløsning. </li><li> Når den første kanal er tilfredsstillende, indleder en anden sekvens for sig selvindirekte, speciel scanning. Generelt er det bedst at scanne længere bølgelængde fluorofor først. Gentag trin 6.1 på anden kanal. </li></ol></li><li> Bekræft mangel på spektral overlapning af imaging enkelt farvede kontrol med både scan sekvenser. Mild spektral overlapning kan korrigeres ved hjælp af spektrale un-blanding funktioner i softwaren, bør dog så vidt muligt undgås. </li></ol> 7.. Image Acquisition <ol><li> Anskaf billeder. Til kvantitativ billeddannelse, anbefales det at opnå mindst 20 billeder / tilstand. Det præcise antal, men bør defineres i henhold til den eksperimentelle spørgsmål i samråd med en statistisk metode, såsom beregning stikprøvestørrelse. Gem eksperiment. </li></ol> 8.. Deconvolution <ol><li> Åbn fil med deconvolution software eller batch-processor. Tjek parametre for hver kanal ved hjælp af software. Bekræft hver kanals excitation og emission spektre STED udtømning emission, og billedbehandling retningning (op eller ned, hvis billedet er 3-dimensionel) i særdeleshed. </li><li> Deconvolve bruge deconvolution guiden. Standardindstillingerne er generelt tilstrækkelig, men signal til støjforhold (SNTR) til vil variere fra fluoroforen til fluoroforen og skal bestemmes for hver kanal og hvert eksperiment individuelt. </li></ol>

<ol>
	<li>Stinchcombe, J. C., Bossi, G., Booth, S., Griffiths, G. 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+of+CTL+contains+a+secretory+domain+and+membrane+bridges.">The immunological synapse of CTL contains a secretory domain and membrane bridges.</a> Immunity. 15 (5), 751-761 (2001).</li><li>Grakoui, A., Bromley, S. K., Sumen, C., 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=The+immunological+synapse:+a+molecular+machine+controlling+T+cell+activation.">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285 (5425), 221-227 (1999).</li><li>Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N., Kupfer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395 (6697), 82-86 (1998).</li><li>Orange, J. S., Harris, K. E., Andzelm, M. M., Valter, M. M., Geha, R. S., Strominger, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mature+activating+natural+killer+cell+immunologic+synapse+is+formed+in+distinct+stages.">The mature activating natural killer cell immunologic synapse is formed in distinct stages.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (24), 14151-14156 (2003).</li><li>Treanor, B., Lanigan, P. M., Kumar, S., 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=Microclusters+of+inhibitory+killer+immunoglobulin-like+receptor+signaling+at+natural+killer+cell+immunological+synapses.">Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses.</a> J. Cell Biol. 174 (1), 153-161 (2006).</li><li>Abeyweera, T. P., Merino, E., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+signaling+blocks+activating+receptor+clustering+and+induces+cytoskeletal+retraction+in+natural+killer+cells.">Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.</a> J. Cell Biol. 192 (4), 675-690 (2011).</li><li>Beemiller, P., Jacobelli, J., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+the+movement+of+signaling+microclusters+with+cellular+motility+in+immunological+synapses.">Integration of the movement of signaling microclusters with cellular motility in immunological synapses.</a> Nat. Immunol. 13 (8), 787-795 (2012).</li><li>Campi, G., Varma, R., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+agonist+MHC-peptide+complex-dependent+T+cell+receptor+microclusters+as+scaffolds+for+signaling.">Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.</a> J. Exp. Med. 202 (8), 1031-1036 (2005).</li><li>Mossman, K. D., Campi, G., Groves, J. T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+TCR+signaling+from+geometrically+repatterned+immunological+synapses.">Altered TCR signaling from geometrically repatterned immunological synapses.</a> Science. 310 (5751), 1191-1193 (2005).</li><li>Varma, R., Campi, G., Yokosuka, T., Saito, T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster.">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25 (1), 117-127 (2006).</li><li>Yokosuka, T., Sakata-Sogawa, K., Kobayashi, W., 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=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat. Immunol. 6 (12), 1253-1262 (2005).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annu. Rev. Cell. Dev. Biol. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> J. Cell Biol. 190 (2), 165-175 (2010).</li><li>Huang, B., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+fluorescence+microscopy.">Super-resolution fluorescence microscopy.</a> Annu. Rev. Biochem. 78, 993-1016 (2009).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+views+of+the+human+NK+cell+immunological+synapse:+recent+advances+enabled+by+super-+and+high-resolution+imaging+techniques.">New views of the human NK cell immunological synapse: recent advances enabled by super- and high-resolution imaging techniques.</a> Front. Immunol. 3, 421 (2012).</li><li>Hell, S. W., Wichmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt. Lett. 19 (11), 780-782 (1994).</li><li>Klar, T. A., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+resolution+in+far-field+fluorescence+microscopy.">Subdiffraction resolution in far-field fluorescence microscopy.</a> Opt. Lett. 24 (14), 954-956 (1999).</li><li>Klar, T. A., Jakobs, S., Dyba, M., Egner, A., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+with+diffraction+resolution+barrier+broken+by+stimulated+emission.">Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (15), 8206-8210 (2000).</li><li>Rankin, B. R., Moneron, G., Wurm, C. 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=Nanoscopy+in+a+living+multicellular+organism+expressing+GFP.">Nanoscopy in a living multicellular organism expressing GFP.</a> Biophys. J. 100 (12), 63-65 (2011).</li><li>Willig, K. I., Kellner, R. R., Medda, R., Hein, B., Jakobs, S., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+resolution+in+GFP-based+microscopy.">Nanoscale resolution in GFP-based microscopy.</a> Nat. Methods. 3 (9), 721-723 (2006).</li><li>Rak, G. D., Mace, E. M., Banerjee, P. P., Svitkina, T., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+cell+lytic+granule+secretion+occurs+through+a+pervasive+actin+network+at+the+immune+synapse.">Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse.</a> PLoS Biol. 9 (9), (2011).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+channel+STED+nanoscopy+of+lytic+granules+on+actin+filaments+in+natural+killer+cells.">Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells.</a> Commun. Integr. Biol. 5 (2), 184-186 (2012).</li><li>Singh, H., Lu, R., Rodriguez, P. 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=Visualization+and+quantification+of+cardiac+mitochondrial+protein+clusters+with+STED+microscopy.">Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy.</a> Mitochondrion. 12 (2), 230-236 (2012).</li><li>Tonnesen, J., Nagerl UV, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+imaging+for+neuroscience.">Superresolution imaging for neuroscience.</a> Exp. Neurol. 242, 33-40 (2013).</li><li>Kempf, C., Staudt, T., Bingen, 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=Tissue+multicolor+STED+nanoscopy+of+presynaptic+proteins+in+the+calyx+of+held.">Tissue multicolor STED nanoscopy of presynaptic proteins in the calyx of held.</a> PLoS One. 8 (4), (2013).</li><li>Jans, D. C., Wurm, C. A., Riedel, D., 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=STED+super-resolution+microscopy+reveals+an+array+of+MINOS+clusters+along+human+mitochondria.">STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (22), 8936-8941 (2013).</li><li>Gustafsson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surpassing+the+lateral+resolution+limit+by+a+factor+of+two+using+structured+illumination+microscopy.">Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.</a> J. Microsc. 198 (2), 82-87 (2000).</li><li>Gustafsson, M. G., Shao, L., Carlton, P. 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=Three-dimensional+resolution+doubling+in+wide-field+fluorescence+microscopy+by+structured+illumination).">Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination).</a> Biophys. J. 94 (12), 4957-4970 (2008).</li><li>Schermelleh, L., Carlton, P. M., Haase, S., 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=Subdiffraction+multicolor+imaging+of+the+nuclear+periphery+with+3D+structured+illumination+microscopy.">Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy.</a> Science. 320 (5881), 1332-1336 (2008).</li><li>Betzig, E., Patterson, G. H., Sougrat, R., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 13 (5793), 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, 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=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91 (11), 4258-4272 (2006).</li><li>Rust, M. J., Bates, M., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-diffraction-limit+imaging+by+stochastic+optical+reconstruction+microscopy.">Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy.</a> Nat. Methods. 3 (10), 793-795 (2006).</li><li>Banerjee, P. P., Pandey, R., Zheng, R., Suhoski, ., Monaco-Shawver, L., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42-interacting+protein-4+functionally+links+actin+and+microtubule+networks+at+the+cytolytic+NK+cell+immunological.">Cdc42-interacting protein-4 functionally links actin and microtubule networks at the cytolytic NK cell immunological.</a> J. Exp. Med. 204 (10), 2305-2320 (2007).</li><li>Vicidomini, G., Moneron, G., Han, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharper+low-power+STED+nanoscopy+by+time+gating.">Sharper low-power STED nanoscopy by time gating.</a> Nat. Methods. 8 (7), 571-573 (2011).</li></ol>

Forfatterne erklærer, at de ikke har nogen konkurrerende finansielle interesser. Delvis offentliggørelse omkostninger ved denne artikel blev afholdt af Leica Microsystems, Inc.

Forbedringen i opløsning i løbet af konfokal vil være noget afhængig af faktorer, som ikke kan kontrolleres. Disse faktorer omfatter mindre afvigelser i dækglas tykkelse og uoverensstemmelser i montering medier. Det er vigtigt at holde temperaturen og luftfugtigheden i imaging rum så konsekvent som muligt, og det place stråle skal udrettet cirka hver 60 min. Som nævnt i Procedures, skal brug af Vectashield montage medium undgås, da det ikke er foreneligt med STED. Ud over at der, bør man altid bruge # 1.5 dækglas, og hvis den er tilgængelig, kan du bruge dem, der er blevet verificeret til en bestemt tykkelse. En ændring af den fremgangsmåde, der beskrives her, er at billedet yderligere kanaler i konfokal, ved hjælp fluorophores der udleder ved en længere bølgelængde end place stråle. På denne måde kan man billede op til fire kanaler (to i konfokal, to i STED). Hvis du tager denne fremgangsmåde er imidlertid de kanaler med fluoroforer eenhed overfører over STED udtømning laser skal blive afbildet først, da anvendelsen af ​​place stråle vil nedbryder fotoner i disse kanaler. En fordel til denne teknik er anvendelse af tid gating, som også vil forbedre opløsning i konfokal ved at eliminere emission fra fotoner med korte levetider 34. Især vil anvendelse af tid gating, timingen af ​​emissions-detektorer til at korrespondere med pulserende excitation STED, mindske baggrundsfluorescens fra refleksion fra dækglas glas når billeddannelse tæt på den. Selv i et eksperiment ikke er egnet til STED, hvis du bruger en pulserende excitation kilde, kan tid gating være et nyttigt redskab til at forbedre opløsning i konfokal. Der er forskellige ændringer, der kan udnyttes til at forbedre opløsningen i STED. Den ene er at formindske størrelsen af ​​knappenålshullet fra standard 1 Airy enhed, selv om dette også vil reducere mængden af ​​lys, der når prøven. Dette kan der kompenseres for ved at øge lasis magt eller gevinst. En anden er at øge linje gennemsnit, hvilket vil øge mængden af ​​oplysninger, der indsamles for hver foton, forbedre opløsning. Igen, dette kan dog være på bekostning af fotoblegning af prøven, så en balance vil være nødvendigt at balance mellem opløsning og blegning. Ligeledes vil anvendelsen af ​​fluorescerende proteiner, såsom GFP kræver omhyggelig optimering for at undgå blegning. Dette kan opnås ved om nødvendigt faldende STED lasereffekt. Længere tidspunkter vil også give mulighed for større foton nyttiggørelse og reducere blegning. Korrektion for fotoblegning skal der redegøres for, når man analyserer levende STED. Selvfølgelig, billedbehandling i 3 dimensioner i STED er også muligt, og det vil også give en forbedring i forhold til konventionelle konfokal billeddannelse. Dette er især tilfældet, hvis det sker i kombination med deconvolution, selv om der bør udvises forsigtighed for at korrigere for afdrift, der opstår under billeddannelse af flere planer i z-aksen. Hvis du bruger Huygens-softwareat deconvolve er denne korrektion opnås ved hjælp af &quot;stabilisere billedet&quot; funktionen. Med denne fremgangsmåde vil opløsning i z-aksen skal forbedres. Dette er en stor forbedring i forhold til konventionel konfokal billedbehandling, der har dårlig aksial opløsning, og selv i løbet af blot børsnoterede selv, som også har en relativt dårlig z-akse opløsning. Mens erhverve flere stakke i STED, skal der drages omsorg for at undgå blegning af prøven, og om nødvendigt kan man reducere linje gennemsnitsberegning eller laser magt intensitet for at gøre det. Igen skal det bemærkes, at hvis billeddannelse andre fluorophores, der ikke er egnet til STED, at anvendelsen af ​​nedbrydningen bjælke i første sekventiel scanning forhindre emission fra disse kanaler. Derfor er en blandet STED / konfokal tilgang (ved brug af konfokal scanning i kanaler, der udsender ved en bølgelængde på mere end 592 nm) vil desværre ikke være egnet til 3D. For at opsummere, har vi valgt STED som en tilgang på grund af sin relativt let application og forbedring i opløsning i forhold til standard konfokal billeddannelse. Til billeddannelse immun synapse, har det vist en effektiv og værdifuld teknik, der giver os mulighed for at se detaljer i F-actin arkitektur ikke muligt på opløsning over 200 nm. Mens mange af disse detaljer synes subtile, kan de få en dybtgående indvirkning på NK-celle funktion. Således anvender vi den nyeste nanoskopiske imaging-teknologi til tilvejebringelse af oplysninger, der er afgørende for at opretholde menneskers sundhed.

Den immunologiske synapse er et komplekst miljø af signalproteiner og cytoskeletale elementer. Den cytolytiske synapse blev oprindeligt beskrevet som havende en &quot;bulls-eye&quot;-lignende struktur med en ring af actin og adhæsionsmolekyler omkring en central sekretorisk domæne 1-4. Men vi ved nu, at den består af mikroskopiske domæner af aktiv signalering, der kræver kontinuerlig dynamisk cytoskeletal reorganisering for funktion 5-11. Meget af den information, som vi nu har om synapse er afledt af mikroskopi, og immunologer har været early adopters af cutting-edge imaging-teknologi. En sådan ny teknologi er super opløsning mikroskopi. Konventionel lysmikroskopi rumligt begrænset af diffraktion barriere af lys, som sætter den nedre grænse for opløsning for alle fluorescensmikroskopi herunder konfokal på cirka 200 nm. I de seneste år har adskillige teknikker været developed, der tillader opløsning under diffraktion barriere. Disse omfatter stimulation emission udtynding mikroskopi (STED), struktureret belysning mikroskopi (SIM), stokastisk løst mikroskopi (STORM), og fotoaktiverbart lysmikroskopi (PALM). Disse teknikker er blevet revideret i detaljer andetsteds 12-15, men er skitseret nedenfor. Subdiffraction begrænset opløsning genereres i unikke måder i hvert system. Udvælgelsen af ​​en super-opløsning teknik bør derfor være dikteret af eksperimentet og eksperimentelle system af interesse. STED super opløsning opnås ved hjælp af en høj intensitet torroidal udtømning stråle, der selektivt &quot;tavshed&quot; fluorescens omkring hver fluorfor af interesse efter excitation, hvilket resulterer i subdiffraction begrænset fluorescensmikroskopi 16-18. En fordel ved STED er, at billedet erhvervelse er hurtig og kræver forholdsvis lidt efterbehandling. Mens farvestof udvælgelse er dikteret af spectral position udtømning bjælke, som i det kommercielt tilgængelige system er beliggende ved 592 nm, er flere kommercielt tilgængelige farvestoffer rådighed, der gør kombinationer af to fluorophorer muligt. Endvidere kan almindeligt anvendte fluorescerende reportere såsom GFP skal afbildes, hvilket gør levende celle eksperimenter muligt 19,20. Vi har tidligere brugt STED at identificere og kvantificere regioner af F-actin hypodensity der er udnyttet af NK-celler til degranulering 21,22. Vi foreslår, at STED er et godt valg til afbildning af immune synapse på grund af sin relativt fleksibelt tilgængelighed af fluoroforer og overlegen forbedring i opløsning i xy akse. Desuden, på kommercielt tilgængelige STED benyttet til disse forsøg, anvendelse af en high-speed (12.000 Hz) resonans scanner tillader hurtig erhvervelse af billeder med minimal skade på prøver. Begrænset fleksibilitet i farve valg betragtes som en ulempe STED 12,dog dobbelt farve STED er forholdsvis ligetil med flere kommercielt tilgængelige fluorophores. Integrationen af ​​STED med en laser konfokal mikroskop giver også mulighed for yderligere konfokal billeddannelse i kombination med STED, så mens STED er begrænset til to kanaler, kan yderligere strukturer filmede i konfokal med opløsning på ca 200 nm (E. Mace, upublicerede observationer ). Mens vi beskriver anvendelsen af STED til billeddannelse af immunceller, er denne teknologi anvendes til en række celletyper, herunder neurale celler og til visualisering af en bred vifte af cellestrukturer 23-26. SIM anvender en anden tilgang til at generere subdiffraction begrænset billeder. Ved at visualisere kendte periodiske excitation mønstre kan information derefter opnås om den ukendte struktur blive undersøgt efter matematisk transformation 27. Dette giver en stigning i beslutning til ~ 100 nm sideværts 28,29. Fordelen ved SIM-kort er, at det is kompatibel med alle standard konfokal farvestoffer og sonder, men ulempen er, at det er meget langsommere til at hente billeder og disse kræver langvarig efterbehandling 12. Dette begrænser også dets anvendelse til levende celler. Endelig kan billederne super-opløsning genereres ved stokastisk foto-kobling af fluorophores. Denne tilgang er udnyttet i foto-aktiverede lokalisering mikroskopi (PALM) og stokastiske optisk genopbygning mikroskopi (STORM). Ved at scanne flere kamera rammer og lokalisere tilfældigt aktiverede molekyler, der er slået &quot;til&quot; og &quot;off&quot; over tid, er billeder med 20-30 nm opløsning genereres fra akkumulerede rammer 30-32. Det trade-off for denne beslutning er den tid, der kræves for at hente billeder. Her viser vi, i detaljer, protokollen for at forberede og billeddannelse dual farveprøver i STED. I dette system eksitation med en pulserende, afstemmelige, hvidt lys laser. På grund af karakterenaf det pulserende exciteringsstrålen er tid gating for afsløring muliggjort og yderligere stigninger opløsning. Desuden er systemet udstyret med gadolinium hybrid (hydr) detektorer, som er mere følsomme end konventionelle lysforstærkningsrør, således at lavere laser strømkrav. Nedbrydningen stråle til STED anvendes løbende og er tunet til 592 nm, hvilket vil diktere valget af farvestoffer til rådighed for to farver STED. Almindeligt anvendte farvestof kombinationer generelt omfatter en overgearet ved 488 nm (f.eks Alexa Fluor 488, Oregon Green, DyLights grøn eller Chromeo 488) og en overgearet ved 458 nm (såsom Pacific Orange eller Horizon V500). Medens påvisning af de to farvestoffer vil være i en række lignende (og begge er tilgængelige ved nedbrydningen laser), vil excitation forekomme med forskellige bølgelængder. Med et justerbart hvidt lys laser og justerbare detektorer, der maksimere signal, mens fjerne spektral overlapning lavet ret nemt. Som sådan har vi haft god succes med kombinioner af kommercielt tilgængelige farvestoffer, såsom Pacific Orange og Alexa Fluor 488 (her anvendt). Vores protokol er skræddersyet til og beskriver evalueringen af ​​humane NK-celler som repræsenterer historiske fokus for vores laboratorium. Vi er specielt udnytte NK92 cellelinje i dette eksempel, da det er en vi har regelmæssigt anvendt i vores eksperimentelle arbejde 21,33.

<table border="1" cellpadding="0" cellspacing="0" style="width:602px;" width="602">
	<tbody>
		<tr>
			<td style="width:230px;">#1.5 cover slips</td>
			<td style="width:222px;">VWR</td>
			<td style="width:150px;">48393-172</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">BD Cytofix/Cytoperm</td>
			<td style="width:222px;">BD Biosciences</td>
			<td style="width:150px;">554722</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Bovine serum albumin</td>
			<td style="width:222px;">Sigma</td>
			<td style="width:150px;">A2153</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Cotton tipped applicator</td>
			<td style="width:222px;">Fisher Scientific</td>
			<td style="width:150px;">S450941</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Falcon centrifuge tubes (50 ml)</td>
			<td style="width:222px;">VWR</td>
			<td style="width:150px;">352070</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Fetal calf serum (FCS) (500 mL)</td>
			<td style="width:222px;">Atlantic Biologicals</td>
			<td style="width:150px;">S11050</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Goat anti-rabbit Pacific Orange</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">P31584</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Laboratory tissue wipers</td>
			<td style="width:222px;">VWR</td>
			<td style="width:150px;">82003-820</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Nail polish</td>
			<td style="width:222px;">VWR</td>
			<td style="width:150px;">100491-940</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">NK-92 cells</td>
			<td style="width:222px;">ATCC</td>
			<td style="width:150px;">CRL-2407</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Phalloidin Alexa Fluor 488</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">A12379</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Phosphate buffered saline</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">14190250</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Prolong anti-fade reagent</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">P7481</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Purified anti-CD18</td>
			<td style="width:222px;">Biolegend</td>
			<td style="width:150px;">301202</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Purified anti-NKp30</td>
			<td style="width:222px;">Biolegend</td>
			<td style="width:150px;">325202</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Purified anti-perforin</td>
			<td style="width:222px;">Biolegend</td>
			<td style="width:150px;">308102</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">RPMI 1640 medium (500 mL)</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Saponin from Quillaja bark</td>
			<td style="width:222px;">Sigma</td>
			<td style="width:150px;">S4521</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Super PAP pen</td>
			<td style="width:222px;">Life Technologies</td>
			<td style="width:150px;">008899</td>
			<td></td>
		</tr>
		<tr>
			<td style="width:230px;">Triton X-100</td>
			<td style="width:222px;">Electron Microscopy Sciences</td>
			<td style="width:150px;">22142</td>
			<td></td>
		</tr>
		<tr>
			<td style="height:27px;">Material Name</td>
			<td style="height:27px;">Company</td>
			<td style="height:27px;">Catalogue Number</td>
			<td style="height:27px;">Comments (optional)</td>
		</tr>
		<tr>
			<td style="height:27px;">Huygens deconvolution software</td>
			<td style="height:27px;">SVI</td>
			<td style="height:27px;">Contact company</td>
			<td style="height:27px;"></td>
		</tr>
		<tr>
			<td style="height:27px;">Leica SP8 TCS STED microscope</td>
			<td style="height:27px;">Leica Microsystems</td>
			<td style="height:27px;">Contact company</td>
			<td style="height:27px;"></td>
		</tr>
	</tbody>
</table>

visualization of the immunological synapse by dual color time-gated stimulated emission depletion (sted) nanoscopy

Naturlige dræberceller danner stramt reguleret, fintunet immunologiske synapser (IS) for at lysere virusinficerede eller tumorigene celler. Dynamisk aktin reorganisering er afgørende for funktionen af ​​NK-celler og dannelsen af ​​IS. Billeddannelse af F-aktin i synapsen har traditionelt anvendt konfokal mikroskopi, men diffraktion grænsen af ​​lys begrænser beslutning af fluorescensmikroskopi, herunder konfokal til ca 200 nm. Nylige fremskridt inden for imaging-teknologi har muliggjort udviklingen af ​​subdiffraction begrænset super-opløsning billeddannelse. For at visualisere F-actin arkitektur på IS rekapitulere vi NK-celle cytotoksisk synapse ved at overholde NK-celler til at aktivere receptoren på glas. Vi derefter billedet proteiner af interesse ved hjælp af to-farve stimuleret emission udtynding mikroskopi (STED). Dette resulterer i &lt;80 nm opløsning på synapser. Heri beskrives de trin i prøveforberedelse og erhvervelse af billeder ved hjælp af dobbelt coleller STED Nanoscopy at visualisere F-actin på NK IS. Vi illustrerer også optimering af indsamling af prøven ved hjælp af Leica SP8 software og tid-gated STED. Endelig udnytter vi Huygens software til efterbehandling deconvolution billeder.

Er klart, at et primært mål for super-opløsning billeddannelse være en forbedring i forhold til konventionel konfokal mikroskopi. Der er dog nogle almindelige faldgruber, som kan føre til suboptimal opløsning. Disse kræver, at hvert eksperiment optimeres individuelt. I vores repræsentativt eksperiment vi billeddannelse af F-actin-netværket i en NK-celle aktiveret af antistof bundet til glas. Almindelige årsager til (og korrektioner for) en mangel på bedre opløsning på STED løbet konfokal er som følger: <ol><li> Under-prøvetagning (figur 1a). Kan dette føre til kornethed og tab af pixel oplysninger, som fremgår af dårlig opløsning af F-actin filamenter. Øget linje eller ramme gennemsnitsberegning kan ofte rette dette. </li><li> Blegning og / eller over-prøvetagning (figur 1b). Dette kan være forårsaget af langvarig pixel opholdstid som følge af overdreven linje gennemsnit. Alternativt kan det være et resultat af over-scanningen af ​​billedet inden erhvervelse, herunder overforbrug afudtømningen laser. Dette medfører ofte en diset eller slørede billeder. Dette kan korrigeres ved at scanne interesseområde kun minimalt før erhverve eller, hvis det er muligt, at øge laser scanningshastighed. Hvis problemet fortsætter, kan nedbrydningen laser magt reduceres. </li></ol> Ved at opnå den rette balance af pixel opholdstid, magnetisering laser magt, og udtømningen laser magt, et billede med forbedret opløsning og tilstrækkelige oplysninger kan genereres (figur 1c). Opløsningen kan forbedres yderligere ved anvendelse af dekonvolvering (figur 1d). Når købet er optimeret, vil deconvolution forbedre opløsning både kvalitativt og kvantitativt og sub-100 nm opløsning bør rutinemæssigt opnåelige. <img alt="Figur 1" fo:content-width="5in" fo:src="/files/ftp_upload/51100/51100fig1highres.jpg" src="/files/ftp_upload/51100/51100fig1.jpg" /> Figur 1.. Optimering af indkøb og fælles faldgruber STED billeddannelse. NK92 celler blev aktiveret på anti-CD18 og-NKp30 belagt glas i 20 min derefter fikseret, permeabiliseret og farvet for F-actin med phalloidin Alexa Fluor 488. A) Et eksempel på tab af billedinformation grund under-sampling. b) Et eksempel på tab af opløsning på grund af blegning / over-sampling c) betingelser optimeret D) optimerede forhold kan føre til større forbedring i opløsning med deconvolution. Scale bar = 5 um.

Watch this Scientific Journal Video about Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy at JoVE.com

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Her illustrerer vi protokollen for billeddannelse med tofarvede STED Nanoscopy den cytotoksiske immun synapse af NK-celler sammenfattet på glas. Ved hjælp af denne metode, vi får sub-100 nm opløsning på synapse proteiner og cytoskelettet.

Visualiseringen af ​​det immunologiske Synapse med Dual Color Time-gated stimuleret emission Nedbrydning (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

visualization-immunological-synapse-dual-color-time-gated-stimulated

Research

JoVE Journal

Immunology and Infection

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Udtømning og Rekonstituering af makrofager i mus

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Makrofag Kolesterol Udtømmelse og dens indflydelse på fagocytose af<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-resolution Imaging af Natural Killer Cell Immunologisk Synapse på et glas-understøttet Planar lipiddobbeltlaget

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

En Endothelial Planar Cell Model for Imaging Immunologisk Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

Kvalitativ og kvantitativ analyse af den immun Synapse i det menneskelige System, ved hjælp af Imaging flowcytometri

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualisere aktin og mikrotubulus Cytoskeletons på B-celle immun Synapse ved hjælp af stimuleret Emission udtynding (STED) mikroskopi

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Undersøgelse af organelle dynamik i B-celler under dannelse af immun synapse

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Målrettet antistof blokering ved et dobbelt funktionelt konjugat af antigene peptid og FC-III-Mimetika (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

Visualiseringen af ​​det immunologiske Synapse med Dual Color Time-gated stimuleret emission Nedbrydning (STED) Nanoscopy

Visualiseringen af det immunologiske Synapse med Dual Color Time-gated stimuleret emission Nedbrydning (STED) Nanoscopy