Forfatterne takker Dr. Julia Heim, Walter Ehrlichmann, Ramona Stumm, Funda Cay, Daniel Bukala, Maren Harant samt Natalie Altmeyer for støtte under eksperimenter og dataanalyse. Dette arbeid ble støttet av Werner Siemens-Foundation, DFG gjennom SFB685 (prosjekt B6) og Fortune (2309-0-0).

Sikkerhets forholdsregler: Ved håndtering av radioaktivitet, lagre 64 Cu bak to-tommers tykt bly klosser og bruk respektive skjerming for alle fartøyer som frakter aktivitet. Bruk passende verktøy for indirekte å håndtere uskjermede kilder for å unngå direkte hudkontakt og minimalisere eksponering for radioaktivitet. Bruk alltid strålingsdosimetri overvåkingsskilt og personlig verneutstyr og sjekk selv og arbeidsområdet for forurensning til umiddelbart å løse det. Kast eventuelt forurenset personl...

<ol>
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Denne protokollen gir en pålitelig og enkel metode for stabilt å radiomerke celler for in vivo sporing av PET. Ved hjelp av denne metode, Cova-TH1-celler, isolert og ekspandert in vitro fra donormus, kan merkes radioaktivt med 64 Cu-DOTA-KJ1-26-mAb og deres homing ble sporet til lungene og perithymic LNS som områder av Cova presentasjon i en Cova-fremkalt akutt luftveis DTHR. Modifiseringen av mAb med chelatoren krever hurtig og effektivt arbeid og bruken av u...

Ikke-invasiv celle-sporing er en allsidig verktøy for å overvåke cellefunksjon, migrasjon og homing in vivo. Nyere celle sporing studier har fokusert på mesenchymale 1, 2 eller benmarg avledede stamceller 3 i sammenheng med regenerativ medisin, autologe perifere hvite blodceller i betennelse eller T-lymfocytter in adoptive cellen terapi mot kreft 3, 4. Klarlegging av områder av handling og de underliggende biologiske prinsipper for celle-baserte terapier er av enorm betydning. CD8 + cytotoksiske T-lymfocytter, genetisk konstruert kimære antigen-reseptor (CAR) T-celler eller tumor-infiltrerende lymfocytter (Tīlss) ble ansett som gullstandarden. Imidlertid har tumor-assosierte antigen-spesifikke TH1-celler vist seg å være et effektivt behandlingsalternativ 4, &lt;/ sup&gt; 5, 6, 7. Som sentrale aktører i inflammasjon, organspesifikke autoimmune sykdommer (for eksempel revmatoid artritt eller astma bronkiale), og celler av høy interesse i cancer immunoterapi, er det viktig å karakterisere de temporale fordeling og homing mønstre av TH1-celler. Noninvasive in vivo avbildning av PET presenterer en kvantitativ, meget følsom fremgangsmåte 8 for å undersøke cellemigreringsmønstre, in vivo homing, og de områder av T-celle responser og handling i løpet av betennelser, allergier, infeksjoner eller tumor avvisning 9, 10, 11. Klinisk, 111 In-oksin brukes for leukocytt-scintigrafi for diskriminering av betennelse og infeksjon 12, mens 2-deoksy-2- (18F) fluor-D-glukose (18F-FDG) blir ofte brukt for celle sporing studier av PET-3, 13. En stor ulempe med denne PET tracer, er imidlertid den korte halveringstid av radionukliden 18 F ved 109,7 minutter, og den lave intracellulære stabilitet som hindrer avbildning på senere tidspunkter etter adoptiv overføring celle. For lengre sikt in vivo-celle sporing studier av PET, men ustabil i cellene, er 64 Cu-PTSM ofte brukt for å spesifikt merke celler 14 og 15 med minimale skadelige effekter på T-celle-levedyktighet og funksjon 16. Denne protokollen beskriver en metode for å redusere uheldige virkninger på cellenes levedyktighet og funksjon ved hjelp av en T-celle-reseptor (TCR) -spesifikk radioaktivt merket mAb. Først, produksjon av radioisotopen 64 Cu, konjugering av mAb KJ1-26 med the chelator DOTA, og den påfølgende 64 Cu-radiomerkings er vist. I et andre trinn, blir den isolasjon og utvidelse av Cova-TH1 celler av DO11.10 donormus og radiomerking med 64 Cu-lastet DOTA-konjugert mAb KJ1-26 (64 Cu-DOTA-KJ1-26) som er beskrevet i detalj. Vurderingen av opptaksverdier og utstrømning av radioaktivitet med en dosekalibratoren og ved y-telling, henholdsvis, så vel som vurdering av virkningene av 64 Cu-radiomerking på cellelevedyktigheten ved trypan blå eksklusjon og funksjonalitet med IFN-y-ELISA presenteres . For ikke-invasiv in vivo celle sporing, er det utløsning av en musemodell for Cova-fremkalt akutt luftveis DTHR og bilde-innhentings av PET / CT etter adoptiv overføring celle beskrevet. Videre kan denne etikettmetoden kan overføres til forskjellige sykdomsmodeller, murine T-celler med forskjellige TCR eller generelle celler av interesse med membranbundne reseptorer eller uttrykk markers bakenforliggende sammenhengende membran skyt 17.

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">HCl, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td style="width:259px;">1.00318</td>
			<td style="width:189px;">64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Methanol, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td>1.06007</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Isopropanol, Suprapur</td>
			<td style="width:227px;">Merck, Darmstadt, Germany</td>
			<td>1.0104</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Pt/Ir (90/10) plate</td>
			<td style="width:227px;">&#214;gussa</td>
			<td style="width:259px;">Custom made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">PEEK chamber</td>
			<td style="width:227px;">&#214;gussa</td>
			<td style="width:259px;">Custom made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">64Ni</td>
			<td style="width:227px;">Chemotrade</td>
			<td style="width:259px;"></td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Polygram SIL G/UV 254 plate</td>
			<td style="width:227px;">Macherey-Nagel</td>
			<td style="width:259px;">805021</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">Ion exchange column</td>
			<td style="width:227px;">BioRad</td>
			<td style="width:259px;">AG1-X8</td>
			<td>64Cu production</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Solid state target system for PETtrace</td>
			<td style="width:227px;">WKL</td>
			<td style="width:259px;">costum made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">64Cu work-up module</td>
			<td style="width:227px;">WKL</td>
			<td style="width:259px;">costum made</td>
			<td>64Cu production</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Dose calibrator</td>
			<td style="width:227px;">Capintec</td>
			<td style="width:259px;">CRC-25R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PETtrace cyclotron</td>
			<td style="width:227px;">General Electric Medical Systems</td>
			<td style="width:259px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DOTA-NHS</td>
			<td style="width:227px;">Macrocyclics</td>
			<td style="width:259px;">B-280</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-cOVA-TCR antibody (KJ1-26)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">Na2HPO4</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">71633</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:292px;">H+ Chelex 100</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">C7901</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Amicon Ultra-15 filter unit</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">UFC910008</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Rotipuran ultrapure water</td>
			<td style="width:227px;">Carl Roth</td>
			<td style="width:259px;">HN68.3</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Ammonium acetate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td>32301</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PBS</td>
			<td style="width:227px;">University Tuebingen</td>
			<td style="width:259px;"></td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Micro Bio-spin P-6 column</td>
			<td style="width:227px;">Bio-Rad Laboratories</td>
			<td style="width:259px;">7326221</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Sodium citrate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">71497</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Cyclone Plus PhosphorImager&#160;</td>
			<td style="width:227px;">Perkin-Elmer</td>
			<td>L2250116</td>
			<td>DOTA-conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DMEM</td>
			<td style="width:227px;">Merck Millipore</td>
			<td>102568</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">FCS</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">S0115/1004B</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Sodium pyruvate</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L0473</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">MEM-amino acids</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">K0293</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">HEPES&#160;</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L 1613</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">&#160;Penicillin/Streptomycin</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">A2212</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">0.05 mM 2-&#946;-mercaptoethanol</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:259px;">M3148</td>
			<td>ingredient for T cell medium&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DO11.10 mice</td>
			<td style="width:227px;"></td>
			<td>in-house breeding</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">DPBS</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:259px;">14190144</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Cell strainer 40 &#181;m&#160;</td>
			<td style="width:227px;">Corning</td>
			<td style="width:259px;">352340</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">ACK Lysing Buffer</td>
			<td style="width:227px;">Lonza</td>
			<td style="width:259px;">10-548E</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">CD4 MicroBeads, mouse</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-097-145</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">QuadroMACS separator</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-090-976</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">LS column</td>
			<td style="width:227px;">Miltenyi Biotech</td>
			<td style="width:259px;">130-042-401</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">anti-CD4 antibody (Gk1.5)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">anti-CD8 antibody (5367.2)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-rat antibody (MAR18.5)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Rabbit complement MA</td>
			<td style="width:227px;">tebu-Bio</td>
			<td style="width:259px;">CL3221</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Anti-IL-4 antibody (11B11)</td>
			<td style="width:227px;"></td>
			<td>Isolated from hybridoma cell culture</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">cOVA 323-339-peptide&#160;</td>
			<td style="width:227px;">EMC-micro-collections</td>
			<td>Custom order</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">CPG1668-oligonucleotides</td>
			<td style="width:227px;">Eurofins MWG Operon</td>
			<td>Custom order</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">IL-2</td>
			<td style="width:227px;">Novartis</td>
			<td>65483-116-07</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">96-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>655180</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">24-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>662160</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">cell culture flask</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>660175</td>
			<td>TH1 cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">48-well plates</td>
			<td style="width:227px;">Greiner&#160;</td>
			<td>677 180</td>
			<td>cell labeling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Gammacell 1000</td>
			<td style="width:227px;">Best Theratronics</td>
			<td>via inquiry&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gulmay RT225</td>
			<td style="width:227px;">Gulmay</td>
			<td>via inquiry&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Trypan blue</td>
			<td style="width:227px;">Merck Millipore</td>
			<td style="width:259px;">L6323</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Mouse IFN-&#947; ELISA</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">558258</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">PE Annexin V Apoptosis Detection Kit&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">559763</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Tube 5 ml</td>
			<td style="width:227px;">Sarstedt</td>
			<td style="width:259px;">55.476</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Round-bottom tubes&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td style="width:259px;">352008</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Wizard &#947;-counter</td>
			<td style="width:227px;">Perkin-Elmer</td>
			<td>2480-0010</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">ELISA Reader MultiscanEX</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td>51118177</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Microscope</td>
			<td style="width:227px;">Leica</td>
			<td>via inquiry&#160;</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">BD LSRII&#160;</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>via inquiry&#160;</td>
			<td>in vitro evaluation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">BALB/c mice</td>
			<td style="width:227px;">Charles River</td>
			<td style="width:259px;">028</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Aluminum gel</td>
			<td style="width:227px;">Serva Electrophoresis</td>
			<td style="width:259px;">12261.01</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Xylazine</td>
			<td style="width:227px;">Bayer HealthCare</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Ketamine</td>
			<td style="width:227px;">Ratiopharm</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Isoflurane</td>
			<td style="width:227px;">CP-Pharma</td>
			<td>Ordered via University hospital</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">30G needle</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>304000</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Syringe</td>
			<td style="width:227px;">BD Biosciences</td>
			<td>11612491</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Capillaries 10 &#181;l</td>
			<td style="width:227px;">VWR</td>
			<td>612-2439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon PET scanner</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;">no longer available</td>
			<td>in vivo cell trafficking, alternative companies: Bruker, Mediso&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon SPECT/CT scanner</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;">no longer available</td>
			<td>in vivo cell trafficking</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:292px;">Inveon Research Workplace</td>
			<td style="width:227px;">Siemens Healthineers</td>
			<td style="width:259px;"></td>
			<td>image analysis, alternative software: Pmod</td>
		</tr>
	</tbody>
</table>

murine lymphocyte labeling by 64cu-antibody receptor targeting for in vivo cell trafficking by pet/ct

Denne protokollen illustrerer fremstilling av 64 Cu og chelatoren konjugering / radiomerking av et monoklonalt antistoff (mAb), etterfulgt av murin lymfocytt cellekultur og 64 Cu-antistoff-reseptor målsøking av cellene. In vitro-evalueringen av den radiomarkør og ikke-invasiv in vivo-celle-sporing i en dyremodell av en luftvei forsinket type hypersensitivitetsreaksjon (DTHR) av PET / CT, er beskrevet. I detalj er konjugering av et mAb med chelatoren 1,4,7,10-tetraazacyklododekan-1,4,7,10-tetraeddiksyre (DOTA) vist. Etter produksjon av radioaktivt 64 Cu, radioaktiv merking av det DOTA-konjugert mAb er beskrevet. Deretter utvidelse av kylling ovalbumin (Cova) -spesifikk CD4 + interferon (IFN) -γ-produserende T-hjelperceller (Cova-TH 1) og påfølgende radiomerking av Cova-TH1-celler er vist. Forskjellige in vitro teknikker er presentert for å evaluere effects av 64 Cu-radiomerking på cellene, slik som bestemmelse av cellelevedyktigheten ved trypan blå eksklusjon, fargingen for apoptose med annexin V for flowcytometri, og vurderingen av funksjonalitet av IFN-γ enzyme-linked immunosorbent assay (ELISA) . Videre er bestemmelsen av det radioaktive opptak inn i cellene og merking stabilitet er beskrevet i detalj. Denne protokollen beskriver videre hvordan man utfører celle sporing studier i en dyremodell for en luftvei DTHR og, derfor, er induksjonen av Cova-fremkalt akutt luftveis DHTR i BALB / c-mus inkludert. Til slutt blir en robust PET / CT-arbeidsflyt herunder bildeinnsamling, ombygging, og analysen presentert. 64 Cu-antistoff-reseptor målretting tilnærming med påfølgende reseptor internalisering gir høy spesifisitet og stabilitet, nedsatt cellulær toksisitet og lav-avgivelseshastigheter i forhold til vanlige PET-tracere for cellemerking, for eksempel 64 Cu-pyruvaldehyde bis (N4-methylthiosemicarbazone) (64 Cu-PTSM). Til slutt, vår tilnærming gjør det mulig ikke-invasiv in vivo celle sporing av PET / CT med et optimalt signal-til-støy-forhold i 48 timer. Denne eksperimentelle fremgangsmåte, kan overføres til forskjellige dyremodeller og celletyper med membran-bundne reseptorer som er internalisert.

Figur 1 oppsummerer merking av Cova-TH1-celler med 64 Cu-DOTA-KJ1-26-mAb og eksperimentell design for in vitro og in vivo studier som omfattes i denne protokollen. <img alt="Figur 1" src="/files/ftp_upload/55270/55270fig1.jpg" /> Figur 1: 64 Cu-DOTA-KJ1-26-mAb etiketteringsprosessen &amp; Experimental Design. (A) Skjematisk represen...

Watch this Scientific Journal Video about Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT at JoVE.com

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Etter fremstillingen av en 64 Cu-modifisert monoklonalt antistoff-binding til et transgen murin T-celle-reseptor, T-celler er radiomerket in vivo, analysert for levedyktighet, funksjonalitet, merking stabilitet og apoptose, og adoptivt overført til mus med en luftveier forsinket-type hypersensitivitet reaksjonen for ikke-invasiv avbildning av positron emisjon tomografi / beregnet tomografi (PET / CT).

Murine Lymfocytter Merking av<sup&gt; 64</sup&gt; Cu-antistoff Reseptor for målretting<em&gt; In Vivo</em&gt; Celletrafikken av PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Research

JoVE Journal

Immunology and Infection

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induksjon av murine tarmbetennelse etter adoptiv overføring av Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høy</sup&gt; T-celler i immundefekte mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Lymfocyttisolasjonsmetoder fra menneskelig hud for fenotypeanalyser og<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Vindu Chamber modell for<em&gt; I Vivo</em&gt; Cell sporing i Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

påvisning av<em&gt; Trypanosoma brucei</em&gt; Variant Surface glykoprotein Bytte av Magnetic Aktivert Cell Sorting og flowcytometri

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Påvisning av enterohemorrhagiske Escherichia Coli kolonisering i Murine vert av ikke-invasiv i Vivo bioluminescens System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Kvantifisere menneskelige Monocyte Chemotaxis In Vitro og Murine lymfocytt menneskehandel Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analyse av 18FDG PET/CT Imaging som et verktøy for studerer Mycobacterium tuberculosis infeksjon og behandling i ikke-menneskelige primater

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Visualisere intracellulær SNARE smugling av fluorescens levetid Imaging mikroskopi

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Vurdering av overføring av lymfocytter i et ex vivo Transmigration system

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...