Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
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Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Protokollerne nedenfor henviser til isolering af mus MoDC, men de overordnede principper der kan gælde andre DC delmængder celler samt. 12 - 16-uge-forhenværende C57Bl/6j mus blev opretholdt i en American Association for akkreditering af Laboratory Animal Care-akkrediteret animalske faciliteten. Alle protokollerne godkendtes ved Stanford University og Tel Aviv Universitet institutionelle Animal Care og brug udvalget.
1. isolering af Tumor forbundet monocyt-afledte DC
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<ol>
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H., 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=Immune+complex-loaded+dendritic+cells+are+superior+to+soluble+immune+complexes+as+antitumor+vaccine.">Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine.</a> J Immunol. 176 (8), 4573-4580 (2006).</li><li>Carmi, 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=Allogeneic+IgG+combined+with+dendritic+cell+stimuli+induce+antitumour+T-cell+immunity.">Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity.</a> Nature. 521 (7550), 99-104 (2015).</li><li>Carmi, 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=Akt+and+SHP-1+are+DC-intrinsic+checkpoints+for+tumor+immunity.">Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity.</a> JCI Insight. 1 (18), e89020 (2016).</li><li>Kenkel, J. 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Betragtning af det store antal DC kræves for vaccinering mus (ca 2-4 x 106 DC pr. en mus), de fleste af vaccination strategier i mus er baseret på dyrkning af DC fra BM og milt efterfulgt af deres ex vivo -aktivering. Men forsøg at aktivere tumor DC i vivo, ved hjælp af de samme betingelser for aktivering af milt og BM DC, ofte har været mislykket i at fremstille effektive immunitet. I de to efterfølgende publikationer, Carmi mfl. har fundet, at blod og tumor MoDC afviger betyd...

Siden deres opdagelse, har dendritiske celler (DC) været fokus på omfattende forskning på grund af deres unikke evne til at vride T-celle differentiering1. Over de sidste mange årtier, har en omfattende forskningsindsats forsøgt at definere de forskellige DC delmængder og deres funktion under tumor progression og immunitet 2. DCs er sammensat af heterogene cellepopulationer, der adskiller sig fra hinanden i deres mønstergenkendelse receptorer, væv distribution, og vandrende og antigen præsentation kapaciteter3,4,5. Sammenlignet med andre DC delmængder, monocyt-afledte DC (MoDC) er langt mere rigelige i tumorer og nemt kan genereres fra cirkulerende eller tumor infiltrerer monocytter6,7. Derfor, mange kliniske forsøg søger at drage fordel af deres relative forekomst er baseret på i vivo og ex vivo manipulation af autologe MoDC for at fremkalde T-celle immunitet 8,9.
På samme måde, DC-baserede vaccination af eksperimentelle tumor modeller kræver 2-3 serie injektioner, 5-7 dages mellemrum, 1-2 x 106 aktiveret DC pulserende med tumor antigener. Derfor, for at opnå dette store antal DC, de fleste mus undersøgelser har primært brugt MoDC kulturperler fra knoglemarv (BM) prækursorer i GM-CSF for 7-9 dage (IL-4 ikke er påkrævet i indstillingen mus)10,11. Ikke desto mindre, givet at GM-CSF knockout mus er samlet normal DC rum 12,13, og givet de blandede befolkninger fremstillet af denne kultur,14 fysiologiske relevansen af disse DC har blevet draget i tvivl.
Alternativt kan DC være rutinemæssigt isoleret fra milt celler. Men DC omfatter kun omkring 0,3-0,8% af samlede milt celler (resulterer i ca 7 x 105 DC/milten), og af disse celler, kun CD103+ DC og MoDC kan vandre tilbage til lymfoid organer. Da MoDCs omfatter ca. 10-15% af milt DC populationer15,16, give de fleste isolation protokoller ca 1 x 105 MoDC pr. milt. Udvidelse af MoDC kan opnås ved at indsprøjte transfekteret B16 celler, der udskiller GM-CSF, hvilket resulterer i en 100-fold stigning i milt MoDC17. Men brug af MoDC til at udvikle DC vacciner er begrænset, da denne procedure ikke kan gøres i mennesker og de opnåede MoDC er allerede stærkt aktiveret.
Ud over at opnå tilstrækkelig antal DC, indebærer en anden udfordring for at udvikle effektive DC vacciner mod autolog kræftceller manglen på tilstrækkelige faresignaler i indstillingen tumor fuldt aktivere DC. Induktion af co-stimulatory signaler er normalt opnås gennem aktivering af mønstergenkendelse receptorer (PRR), eller c-type lektin signaling veje18,19,20,21. En yderligere tilgang for aktivering DC udnytter deres evne til at tage op antigener gennem interaktioner med overflade Fcγ receptorer (FcγR). En række vigtige håndskrifter har vist, at injektion af MoDC fra BM prækursorer aktiveret hos tumor-IgG IC kan forhindre tumorvækst i profylaktisk indstillinger, og kan føre til udryddelse af etablerede tumorer22,23 .
I to nylige papirer opdagede Carmi et al. , at i modsætning til BMDC og milt DC, MoDC fra blodet og tumorer ikke kan reagere på IgG IC uden yderligere stimuli. Dette blev anset for at være på grund af tilstedeværelsen af høje intracellulære niveauer af tyrosin fosfataser regulerer FcγR signalering24,25. Ved at definere et kritisk kontrolpunkt i DC, gav dette arbejde et vigtigt indblik i kravene til vellykket DC-baserede vaccination. Kravet om for ekstra stimuli for at aktiverer FcγR signalering, og formentlig signalering fra andre lektin receptorer udnytter en lignende fosforylering kaskade, understreger således behovet for at undgå priming af DC under deres isolation.
Derfor, denne protokol beskriver isolation af MoDC fra blodet og tumorer, som afviger markant fra BM og milt DC, og fremhæver forholdsregler værd at overveje i løbet af processen.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

Dendritiske celler (DC) er heterogene cellepopulationer, der adskiller sig i deres cellemembran markører, migrationsmønstre og distribution, og i deres antigen præsentation og T-celle aktivering kapacitet. Da de fleste vaccinationer af eksperimentelle tumor modeller kræver millioner af DC, er de meget isoleret fra knoglemarv eller milt. Disse DC varierer dog betydeligt fra blod og tumor DC i deres svar til immunkomplekser (IC), og formentlig til andre Syk-kombineret lektin receptorer. Vigtigere, givet følsomheden af DC til fare-associerede molekyler, tilstedeværelsen af endotoksiner eller antistoffer der bitmapgenkendelse aktivering receptorer i en af de isolere trin kunne resulterer i priming af DC og dermed påvirke parametrene, eller i det mindste dosering, krævede hen til aktivere dem. Derfor, her vi beskrive en detaljeret protokol til at isolere MoDC fra blodet og tumorer samtidig undgå deres for tidlig aktivering. Derudover tilbydes en protokol for MoDC aktivering med tumor IC, og deres efterfølgende analyser.

Vi sammenlignede oprindeligt antistoffer fra naiv syngeneic og allogen mus evne til at binde til tumorceller. Med henblik herpå, blev B16F10 og LMP tumor cellelinjer fast i PARAFORMALDEHYD og vasket grundigt. B16F10 er et melanom cellelinie, som blev oprindeligt isoleret fra lungemetastaser i C57Bl/6 mus. LMP er en pancreas tumor celle, der blev isoleret fra KrasG12D / + LSL-Trp53R172H / + og Pdx-1-Cre mus, og vokser støt i 129F1 mus. For at opnå IC, blev tumorceller udruget i 20 min. ...

Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyt-afledte DC (MoDC) kan føle mindre mængder af fare-associerede molekyler og er derfor let primet. Vi leverer en detaljeret protokol for dyrkning af MoDC fra blodet og tumorer og deres aktivering med immunkomplekser samtidig fremhæve vigtige forholdsregler, der bør overvejes for at undgå deres for tidlig aktivering.

Isolation protokol af musen monocyt-afledte dendritiske celler og deres efterfølgende In Vitro aktivering med Tumor immunkomplekser

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

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Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

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Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

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