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

Protokollene nedenfor refererer til isolering av musen MoDC, men de generelle prinsippene gjelder kanskje andre DC delsett celler, også. 12 - 16-uke-gamle C57Bl/6j mus ble opprettholdt i et American Association for akkreditering av laboratoriet Animal Care-akkreditert dyr anlegget. Alle protokoller ble godkjent av Stanford University og Tel-Aviv University institusjonelle Animal Care og bruk komiteen.
1. isolering av svulst forbundet monocytt-avledet DC
<ol><li>I laminær strømning hette, ...

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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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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+lipopolysaccharide+(LPS)+pretreatment+of+mouse+macrophages+modulates+LPS-dependent+interleukin-6+production+in+vitro.">Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 production in vitro.</a> Infect Immun. 64 (3), 1011-1015 (1996).</li><li>Deng, H., Maitra, U., Morris, M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+responsible+for+the+priming+of+macrophage+activation.">Molecular mechanism responsible for the priming of macrophage activation.</a> J Biol Chem. 288 (6), 3897-3906 (2013).</li><li>Cella, 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=A+novel+inhibitory+receptor+(ILT3)+expressed+on+monocytes,+macrophages,+and+dendritic+cells+involved+in+antigen+processing.">A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing.</a> J Exp Med. 185 (10), 1743-1751 (1997).</li><li>Kramer, P. 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Gitt det store antallet DC kreves for vaccinating mus (ca 2-4 x 106 DC per ett museklikk), de fleste av vaksinasjon strategier i mus er basert på isolering av DC fra BM og milt etterfulgt av ex vivo aktivisering. Imidlertid forsøker å aktivere svulst DC i vivo, bruker de samme betingelsene for å aktivere milten og BM DC, har ofte vært mislykket produsere effektive immunitet. I to påfølgende publikasjoner, Carmi et al. har funnet at blod og tumor MoDC avvike betydelig fra milt ...

Siden oppdagelsen sin, har dendrittiske celler (DC) vært fokus for omfattende forskning unike evne til å skew T celle differensiering1. De siste tiårene, har et omfattende forskning forsøk søkt å definere ulike DC delsettene og deres funksjon under svulst progresjon og immunitet 2. DCs er sammensatt av ulike celle populasjoner som skiller seg fra hverandre i deres mønstergjenkjenning reseptorer, vev distribusjon, trekkfugler og antigen presentasjon evner3,4,5. Sammenlignet med andre DC undergrupper, monocytt-avledet DC (MoDC) er langt mer rikelig i svulster og lett kan genereres fra sirkulerende eller svulst-infiltrere monocytter6,7. Derfor er mange kliniske forsøk søker å utnytte sin relative utbredelse basert på i vivo og ex vivo manipulering av autologous MoDC for å lokke fram T celle immunitet 8,9.
Tilsvarende DC-baserte vaksinasjon av eksperimentelle svulst modeller krever 2-3 føljetong injeksjoner, 5-7 dagers mellomrom, 1-2 x 106 aktivert DC pulserende med svulst antigener. Derfor, for å oppnå antall DC, de fleste mus studier har primært brukes MoDC kultivert fra benmargen (BM) forløpere i GM-CSF 7-9 dager (IL-4 ikke er nødvendig i innstillingen mus)10,11. Likevel, gitt at GM-CSF knockout mus har samlet normal DC kupé 12,13, og gitt blandet befolkningen fått fra denne kulturen,14 fysiologiske relevansen av disse DC har blitt kalt inn spørsmålet.
Alternativt kan DC være rutinemessig isolert fra milt celler. Imidlertid DC utgjør bare 0,3 0,8% av totale milt celler (resulterer i ca 7 x 105 FM/milt), og av disse cellene, bare CD103+ DC og MoDC kan overføre tilbake til lymfoide organer. Siden MoDCs utgjør ca 10-15% av splenic DC bestander15,16, gir de fleste isolasjon protokoller ca 1 x 105 MoDC per milten. Utvidelse av MoDC kan oppnås ved å injisere transfekterte B16 celler det avsondre GM-CSF, resulterer i en 100-fold økning i splenic MoDC17. Men bruk av MoDC for å utvikle DC vaksiner er begrenset ettersom denne fremgangsmåten ikke kan utføres i mennesker og innhentet MoDC er allerede svært aktivert.
I tillegg til å skaffe tilstrekkelig antall DC, innebærer en utfordring for å utvikle effektive DC vaksiner mot autologous kreftceller mangel på tilstrekkelig fare signaler i innstillingen svulst fullt aktivere DC. Induksjon av co-stimulatory signaler er vanligvis oppnås gjennom aktivering av mønstergjenkjenning reseptorer (PRR), eller c-type Lektiner signalnettverk trasé18,19,20,21. En ytterligere tilnærming for å aktivere DC utnytter deres evne til å ta opp antigener gjennom interaksjoner med overflate Fcγ reseptorer (FcγR). Faktisk en rekke viktige manuskripter har vist at injeksjon av MoDC fra BM forløpere aktivert med svulst-IgG IC kan hindre tumor vekst i forebyggende innstillinger, og kan føre til utrydding av etablerte svulster22,23 .
To nyere verk oppdaget Carmi et al. at i motsetning til BMDC og milt DC, MoDC fra blod og svulster ikke kan svare på IgG KI uten ekstra stimuli. Dette ble funnet for å være av høy intracellulær nivåer av tyrosin phosphatases regulerer FcγR signalering24,25. Ved å definere en kritisk checkpoint i DC, gitt dette arbeidet en viktig innsikt i kravene for vellykket DC-baserte vaksinasjon. Behovet for ytterligere stimuli aktivere FcγR signalering, og antagelig signalene fra andre Lektiner reseptorer bruker en lignende fosforylering cascade, dermed understreker behovet for å unngå grunning FM under deres isolasjon.
Derfor nåværende protokollen beskriver isolering av MoDC fra blod og svulster, som avviker markant BM og milt DC, og fremhever forholdsregler verdt å vurdere under prosessen.

<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

Dendrittiske celler (DC) er heterogene celle populasjoner som avviker i cellemembranen markører, migrasjon og distribusjon, og i deres antigen presentasjon og T celle aktivering kapasiteter. Siden de fleste vaksiner eksperimentelle svulst modeller krever millioner av DC, er de mye isolert fra benmarg eller milten. Men disse DC betydelig forskjellig fra blod og svulst DC i sine Svar å immun komplekser (IC) og formodentlig andre forhold-kombinert Lektiner reseptorer. Viktigst, gitt sensitiviteten av DC til fare-assosiert molekyler, endotoxins eller antistoffer som krysskobling aktivisering reseptorer i en av de isolere kan medføre grunning FM og dermed påvirker parametere, eller minst dosering, må aktivere dem. Derfor beskriver her vi en detaljert protokoll for å isolere MoDC fra blod og svulster og unngå deres tidlig aktivering. I tillegg finnes en protokoll for MoDC aktivering med svulst IC og deres påfølgende analyser.

Vi sammenlignet først kapasitet av antistoffer fra naive syngeneic og allogene mus til å binde til kreftceller. Dette ble B16F10 og LMP svulst cellelinjer løst i paraformaldehyde og vasket mye. B16F10 er melanom cellen linje, som ble opprinnelig isolert fra lunge metastaser i C57Bl/6 mus. LMP er en bukspyttkjertelen svulst celle som ble isolert fra KrasG12D / +, LSL-Trp53R172H / + og Pdx-1-grobunn mus, og vokser jevnt i 129F1 mus. For å få IC, ble kreftceller inkubert etter 20 min p?...

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

Monocytt-avledet DC (MoDC) kan forstand mindre mengder fare-assosiert molekyler og er derfor lett fylt. Vi gir en detaljert protokoll for isolering av MoDC blod svulster og Aktivisering med immun komplekser mens uthevingen nøkkel forholdsregler som bør vurderes for å unngå deres tidlig aktivering.

Isolasjon protokollen av musen monocytt-avledet dendrittiske celler og deres påfølgende In Vitro aktivisering med Tumor immun komplekser

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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Flytte oppover: En enkel og fleksibel In Vitro tredimensjonale invasjonen analysen protokoll

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Mesenchymal stamceller isolert fra cellulose vev og co kultur kreftceller å studere deres samhandling

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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Sanntid påvisning av i Vitro svulst celle apoptose indusert av CD8+ T celler å studere immun undertrykkende funksjoner av svulst-infiltrere myeloide celler

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Intramucosal vaksinasjon av Squamous celle Carcinoma celler i mus for svulst immun profilering og behandling respons vurdering

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

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Overføring av manipulert tumor-Associated nøytrofile til tumor-bærende mus for å studere deres angiogenic potensial in vivo

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