Name: investigating mast cell secretory granules; from biosynthesis to exocytosis
Uploaded: 2015-01-26T14:00:00
Description: Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Vi takker Dr. U. Ashery for gaven af ​​NPY-mRFP cDNA. Vi takker Drs. MJ Kofron, L. Mittleman, M. Shaharbani, og Y. Zilberstein for uvurderlig hjælp med mikroskopi og image-analyser. Vi takker også Dr. Joseph Orly til kritisk læsning af dette manuskript. Dette arbejde blev støttet af en bevilling fra Israel Science Foundation, der blev grundlagt af Israel Akademi for Videnskaber (1139-1112 til RS-E.).

1. Fremstilling af RBL cellekulturmedier <ol><li> Bland 500 ml lav glucose Dulbeccos modificerede Eagles medium (DMEM) med 56 ml føtalt bovint serum (dette gør 10% FBS), og derefter tilsættes 5,5 ml penicillin streptomycin (dette gør ~ 1% PEST). </li><li> Filtrer mediet med 500-1.000 ml flaske-Top Vacuum Filtre med 0,22 um porestørrelse og opbevares ved 4 ° C. </li></ol> 2. Kultur af RBL Cells <ol><li> Grow RBL-celler i en fugtig atmosfære af 5% CO2 ved 37 ° C i enten plade...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+proteases+as+protective+and+inflammatory+mediators.">Mast cell proteases as protective and inflammatory mediators.</a> Adv Exp Med Biol. 716, 212-234 (2011).</li><li>Lundequist, A., Pejler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+implications+of+preformed+mast+cell+mediators.">Biological implications of preformed mast cell mediators.</a> Cell Mol Life Sci. 68 (6), 965-975 (2011).</li><li>Metz, M., Maurer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells--key+effector+cells+in+immune+responses.">Mast cells--key effector cells in immune responses.</a> Trends Immunol. 28 (5), 234-241 (2007).</li><li>Gordon, J. R., Burd, P. 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Vi beskriver en innovativ strategi, der kombinerer kvantificering af MCs exocytose og fire (x, y, z, t) dimension kvantificeringer af tidsforkortet tredimensional billeddannelse af SGS i levende celler under anvendelse af et reportergen for exocytose. Denne teknik gør det muligt for screening af familier af proteiner til deres indvirkning på MC-funktion, såsom overvågning generalsekretærerne starter så tidligt som deres exit fra Golgi gennem deres modning, køb af exocytose kompetence og degranulering. Kombination...

Mastceller (MC) er immunceller, der er bedst kendt for deres deltagelse i allergiske og inflammatoriske reaktioner, såsom gigt, astma, eosinofil esophagitis, kronisk dermatitis og anafylaktisk shock 1,2 samt andre patologier, herunder koronararteriesygdom 3,5 og cancer 3,4. Desuden MCs spiller en vigtig rolle i medfødte og adaptive immunitet, både i vært forsvar mod bakterier og parasitter, og ved undertrykkelse af immunreaktioner, fx inducere allograft tolerance 5,6. MC&#39;er stammer fra knoglemarven, udvikling fra CD34 + / CD117 + pluripotente stamceller 7. Committed knoglemarv MC progenitorer frigives til blodbanen og migrere ind i de perifere væv lokalisering overvejende inden bindevæv og epiteloverflader 8. Modning og terminal differentiering til sidst opnås under indflydelse of cytokiner inden det omgivende miljø 8,9. MCs kan aktiveres af et allergen (antigen, Ag), hvis møde resulterede i dannelsen af ​​immunglobulin E (IgE) type antistoffer. Binding af sådanne IgE til MC FceRI receptorer, efterfulgt af krydsbinding af cellebundet IgE ved fornyet behandling med det samme Ag, resulterer i FceRI sammenlægning og indledning af en signaleringskaskade der kulminerer i celle degranulering [revideret i 10,11] . MCs også aktiveres uafhængigt af IgE, som neuropeptider 5,12, toksiner 13, bakterielle og virale antigener 14,15, en række positivt ladede peptider kollektivt benævnt grundlæggende sekretagoger, immunceller og cytokiner 5,13,12,16 , 17. MCs også aktiveres af mange af deres egne frigivne mediatorer, hvilket yderligere forstærker den inflammatoriske respons. MCs er pakket med sekretoriske granulat (SGS), som indeholder immunoregulatoriske mediatorer, herunder vasoaktive aminer, såsom histamin og serotonin (i gnavere), proteoglycaner, proteaser, såsom chymase og tryptase, vaskulær endotelvækstfaktor og flere cytokiner og chemokiner 8,9. Disse mæglere er &quot;klar til at gå&quot;, og når MC&#39;er aktiveres af en passende stimulus, er disse mediatorer frigives fra cellerne ved reguleret exocytose (degranulering) i løbet af få sekunder til minutter 18,19. Denne indledende begivenhed efterfulgt af de novo-syntese og frigivelse af en bred vifte af biologisk potente stoffer, herunder arachidonsyremetabolitter, multiple cytokiner og chemokiner 20,21,22. Frigivelse af nyligt syntetiserede produkter sker uafhængigt af SG udgivelse. Kollektivt, disse mediatorer indlede tidlige og sene fase inflammatoriske og allergiske reaktioner. Derfor forstå de mekanismer, der tegner sig for MC-aktivering og degranulering er både teoretisk og klinisk importance. Vanskeligheden for genetisk manipulere primære og dyrkede MC&#39;er har hæmmet forsøgene på at belyse mekanismerne bag MC degranulering, som forblev dårligt løst. For at overvinde dette problem, vi udviklede en reporter assay ved co-transfektion af mucosale mastcellelinje, rotte basofile leukæmi (RBL) -2H3 (i det følgende benævnt RBL) eller knoglemarv afledte MC&#39;er (BMMCs) 30 med et gen af interesse og neuropeptid Y (NPY) fusioneret til monomere RFP (mRFP) som SG reporter. NPY er tidligere vist at rekapitulere adfærd endogene SG markører i andre systemer. Desuden, på grund mRFP fluorescens pH ufølsom ekspression af NPY-mRFP tillader visualisering af de sure strain gauges samt kvantitativ vurdering af exocytose ved anvendelse af 96-brønds plader og en fluorescenspladelæser. Vi har vist, at NPY-mRFP leveres til de sure generalsekretærerne for RBL celler og BMMCs og frigives fra cellernepå en reguleret måde ved siden af endogene SG last (dvs. β-hexosaminidase og serotonin) 30, 32. Denne protokol giver en høj opløsning billeddannelse-baserede metode, der tillader screening gener af interesse for deres fænotypiske og funktionelle indvirkning på SG egenskaber og degranulering i RBL-celler 32. Konkret denne protokol giver real time tracking af MC SGs og kvantificering af deres område eller volumen størrelse, deres antal, kinetik delene, deres bevægelse langs cellens cytoskelet og deres endelige fusion med plasmamembranen under forskellige forhold. For eksempel, sensibiliserende af cellerne med DNP-specifikt IgE og udløser cellerne med en multivalent Ag (DNP konjugeret serum albumin) under forskellige forstyrrelser (dvs. knockdown af gener af interesse, overekspression af vildtype eller mutante gener eller farmakologiske manipulationer) og sammenligning med kontrolceller.

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	<tbody>
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			<td height="20" style="height:20px;width:421px;">Name of Material/ Equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:156px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:421px;">DMEM</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D6046-500ML</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Fetal Bovine Serum</td>
			<td style="width:128px;">GE health care Life sciences</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td style="width:128px;">Life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Cellulose acetate membrane, pore size 0.22&#160;&#956;m</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430769-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning tissue-culture treated culture dishes</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS430167</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Trypsin/EDTA Solution (TE)</td>
			<td style="width:128px;">Life technologies</td>
			<td>R001100</td>
			<td style="width:156px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">PIPES dipotassium salt</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">108321-27-3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium acetate hydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>114460-21-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium acetate tetrahydrate</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">M5661&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">L-Glutamic acid potassium salt monohydrate (Potassium glutamate)</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 mm electroporation cuvettes</td>
			<td style="width:128px;">cell projects</td>
			<td style="width:139px;">EP-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GENE PULSER WITH PULSE CONTROLLER &#38; CAPACITANCE</td>
			<td style="width:128px;">Bio rad</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chambered coverglass</td>
			<td style="width:128px;">Thermo scientific</td>
			<td align="right" style="width:139px;">155411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">24 well, flat bottom</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Corning 96 well plates</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">CLS3367 or CLS390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well plate fluorescence reader- Infinite 200</td>
			<td style="width:128px;">Tecan</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium ionophore A23187</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td height="20" style="height:20px;width:139px;">C7522</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-O-tetradecanoyl-13-acetate (TPA)</td>
			<td>Calbiochem</td>
			<td height="20" style="height:20px;width:139px;">P3766</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-DNP monoclonal IgE</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td>D8406&#160;</td>
			<td height="20" style="height:20px;width:156px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNP-BSA/ DNP-HAS</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A6661</td>
			<td>Avoid from direct light exposure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Triton-x-100</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Confocal fluorescent microscope:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Zeiss LSM 510</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Leica</td>
			<td style="width:128px;">SP5</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon A1 inverted</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Imaris software</td>
			<td style="width:128px;">BITLANE</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Microsoft exel or Prism or other analyses software</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Other reagent:</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Magnesium Chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5833</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sodium chloride</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">6404</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Calcium chloride&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">2382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin&#160;</td>
			<td style="width:128px;">Sigma-Aldrich</td>
			<td style="width:139px;">A4503</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Glucose</td>
			<td style="width:128px;">BDH Laboratories</td>
			<td style="width:139px;">284515V</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monosodium phosphate&#160;</td>
			<td style="width:128px;">MERK</td>
			<td style="width:139px;">5345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:421px;">Sterile water</td>
			<td style="width:128px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating mast cell secretory granules; from biosynthesis to exocytosis

Mastceller (MC) er sekretoriske celler i immunsystemet, der udføre deres fysiologiske og patologiske funktioner ved at frigive præformede og nyligt syntetiserede allergiske, inflammatoriske og immunregulerende mediatorer. MC&#39;er &#39;mediatorer påvirker flere væv og organer kulminerede i allergiske og immunrespons. Syntesen, opbevaring og frigivelse af MC mæglerne er stærkt reguleret. De præformede mediatorer er pakket i cytoplasmatiske sekretoriske granula (SG), der fusionere med plasmamembranen og frigøre deres indhold ved reguleret exocytose. Vi præsenterer en protokol, baseret på co-ekspression af et gen af ​​interesse med et reportergen, der er målrettet til SGS og frigives på en reguleret måde ved siden af ​​de endogene SG mediatorer. Protokollen giver høj opløsning fire dimensional konfokal analyser af MC SGs og overvåge deres tidslinje fra biogenese til udløst exocytose. Således ved hjælp af denne protokol til screening gener af interest for deres fænotypiske og funktionelle konsekvenser tillader decifrere de molekylære mekanismer, der styrer biogenese og exocytose MC SGs og identificere de involverede myndigheder. Derved bør ydes yderligere indsigt i de cellulære mekanismer, der tegner sig for MC&#39;er funktion i sundhed og sygdom.

På grund af den lave transfektionseffektivitet MCS, er det usandsynligt at efterlade en indvirkning på udlæsninger af gennemsnitlig sekretion målt af endogene SGS mediatorer genetiske manipulationer. Men ved at opnå fuldstændig co-ekspression af reportergenet NPY-mRFP og co-transficeret plasmid på de samme celler, overvågning af NPY-mRFP resulterer i udelukkende at overvåge cellepopulation, der udtrykker genet af interesse. Derfor fordel af dette assay sammenlignet med konventionelle metoder er evnen til selekt...

Watch this Scientific Journal Video about Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis at JoVE.com

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Målet med denne protokol var at udvikle en metode, der vil gøre det muligt for funktionelle genomiske analyser af mast celle sekretion. Protokollen er baseret på kvantitativ vurdering af udgivelsen af ​​en fluorescerende reporter gen cotrasfected med genet af interesse og tidstro analyser af sekretoriske granula morfologi.

Undersøgelse Mast Cell Sekretorisk Granulat; fra Biosyntese til exocytose

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Research

JoVE Journal

Immunology and Infection

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Primære Mikroglia Isolering af blandede gliacelle Kulturer af neonatal rottehjerne Tissue

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Klinisk anvendelse af<em&gt; Tornerose</em&gt; Artificial og antigen-præsenterende celler til genetisk at modificere T-celler fra perifert og navlestrengsblod

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Isolering af precursor B-celle-undergrupper fra navlestrengsblod

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

En Microplade Assay til vurdering af kemiske Virkninger på RBL-2H3 mastcelledegranulering: Effekter af Triclosan uden brug af et organisk opløsningsmiddel

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Visualisere Ikke-lytiske exocytose af<em&gt; Cryptococcus neoformans</em&gt; Fra Makrofager hjælp af digitale lysmikroskopi

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Analyse af mastcellernes funktioner i Vivo Ved hjælp af 'Mast Cell Knock-in' Mus

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overekspression og oprensning af mennesket<em&gt; Cis</em&gt; -prenyltransferase i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

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

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

Isolering af bughinden-afledte mastceller og deres funktionelle karakterisering med Ca2 +-billedbehandling og degranulering 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 ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Undersøgelse af aortaklappen Forkalkning via isolation og kultur af T-lymfocytter ved hjælp af feederceller fra bestrålet Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...