Name: saturated fatty acids induce ceramide-associated macrophage cell death
Uploaded: 2017-10-31T15:00:00
Description: Watch this Scientific Journal Video about Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death at JoVE.com

Dette arbejde blev støttet delvis ved University of Louisville start-up midler og National Cancer Institute (Bethesda, MD) giver R01CA177679, R01CA180986.

protokollen blev godkendt af institutionelle Animal Care og brug udvalg (IACUC) på University of Louisville.
 1. mus knoglemarv afledt makrofager (BMDMs) <ol><li>aflive en 6 til 8 uger gamle, sunde vildtype mus ved hjælp af CO 2. Pin det ned til en skum bord og spray det med 70% ethanol, indtil det er gennemblødt. Fjerne skinneben/lårben ben med saks og pincet. Læg dem i en petriskål med 5 mL 1 x fosfatbufferet saltopløsning (PBS). Udføre alle procedurer under sterile for...

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	<li>Martins de Lima, T., Cury-Boaventura, M. F., Giannocco, G., Nunes, M. T., Curi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+fatty+acids+on+a+macrophage+cell+line+(J774).">Comparative toxicity of fatty acids on a macrophage cell line (J774).</a> Clin Sci (Lond). 111 (5), 307-317 (2006).</li><li>Simard, J. R., Zunszain, P. A., Hamilton, J. A., Curry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+high+and+low+affinity+fatty+acid+binding+sites+on+human+serum+albumin+revealed+by+NMR+drug-competition+analysis.">Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis.</a> J Mol Biol. 361 (2), 336-351 (2006).</li><li>Penn, A. H., Dubick, M. A., Torres Filho, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+Acid+Saturation+of+Albumin+Used+in+Resuscitation+Fluids+Modulates+Cell+Damage+in+Shock:+In+Vitro+Results+Using+a+Novel+Technique+to+Measure+Fatty+Acid+Binding+Capacity.">Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: In Vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.</a> Shock. , (2017).</li><li>Vusse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Albumin+as+fatty+acid+transporter.">Albumin as fatty acid transporter.</a> Drug Metab Pharmacokinet. 24 (4), 300-307 (2009).</li><li>Zhang, 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=Adipose+Fatty+Acid+Binding+Protein+Promotes+Saturated+Fatty+Acid-Induced+Macrophage+Cell+Death+through+Enhancing+Ceramide+Production.">Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-Induced Macrophage Cell Death through Enhancing Ceramide Production.</a> J Immunol. 198 (2), 798-807 (2017).</li><li>Zhang, 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=Epidermal+Fatty+Acid+binding+protein+promotes+skin+inflammation+induced+by+high-fat+diet.">Epidermal Fatty Acid binding protein promotes skin inflammation induced by high-fat diet.</a> Immunity. 42 (5), 953-964 (2015).</li><li>Wen, 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=Fatty+acid-induced+NLRP3-ASC+inflammasome+activation+interferes+with+insulin+signaling.">Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.</a> Nat Immunol. 12 (5), 408-415 (2011).</li><li>Zhang, 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=Fatty+acid-binding+protein+E-FABP+restricts+tumor+growth+by+promoting+IFN-beta+responses+in+tumor-associated+macrophages.">Fatty acid-binding protein E-FABP restricts tumor growth by promoting IFN-beta responses in tumor-associated macrophages.</a> Cancer Res. 74 (11), 2986-2998 (2014).</li><li>Ulloth, J. 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En god forberedelse af fedtsyre løsning er af afgørende betydning at studere den biologiske funktion af fedtsyrer. Neutralisering af fedtsyrer øger dets Opløselighed i vandig opløsning. Natrium salte af fedtsyrer, især mættede fedtsyrer, er dog stadig af lav Opløselighed i vand eller PBS så vi observeret. Én metode er at bruge 95-100% ethanol til at hjælpe opløse fedtsyre1. Brug denne metode, kan højere toksicitet for celler observeres selv ved lavere koncentrationer for mindre giftig...

Fedtsyrer spiller en afgørende rolle i energimetabolisme og i syntesen af membran fosfolipider i forskellige typer af celler. Fedtsyrer har en lav vandig opløselighed. Passende forberedelse af fedtsyre er af afgørende betydning for at studere de biologiske funktioner af fedtsyrer i pattedyrsceller. Når fedtsyrer er tilberedt med ethanol, kan mange fedtsyrer vise deres giftige sæbe (vaskemiddel) effekt på cellemembranen, selv ved relativt lave koncentrationer1. Som en naturlig, store transporter til frie fedtsyrer i serum betragtes serum albumin som en god luftfartsselskab for fedtsyre levering i vitro for fedtsyre funktion assays2,3,4. Men detaljerne i forberedelsen af fedtsyre og serum albumin konjugat er normalt ikke tilgængelig selvom mange forskningspapirer ved hjælp af fedtsyrer er blevet offentliggjort.
Makrofager udtrykke meget epidermal fedtsyre-bindende protein og fedt fedtsyre-bindende protein5,6,7,8. De aktivt optagelse mættede og umættede fedtsyrer, som kan regulere deres immun funktioner. For at studere virkningerne af fedtsyrer på makrofager og andre celler, var forskellige metoder af fedtsyre forberedelse anvendt1,7,9. Ved hjælp af passende rede fedtsyre/serum albumin konjugater for at undersøge virkningen af fedtsyrer på makrofag funktion er af afgørende betydning at opnå biologisk meningsfuld data. Undersøgelser af virkningen af fedtsyrer på makrofag funktion kan give grundlæggende viden og potentielle terapeutiske mål vedrørende fedtsyre metabolisme i makrofag-involveret sygdomme.

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			<td height="20" style="width:357px;height:20px;">CPX Ultrasonic Bath&#160;</td>
			<td style="width:208px;">Bransonic</td>
			<td style="width:153px;">Model 2800</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium palmitate (PA)</td>
			<td>Nu-Chek Prep, Inc.</td>
			<td>S-1109</td>
			<td>M.W. 278</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium stearate (SA)</td>
			<td>Nu-Chek Prep, Inc.</td>
			<td>S-1111</td>
			<td>M.W. 306</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA), fatty acid-free</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse macrophage colony stimulating factor (mM-CSF)&#160;</td>
			<td>Cell Signaling Technology,&#160; Inc.</td>
			<td>5228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>VWR International</td>
			<td>71002-878</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Annexin V, Alexa Fluor 488 conjugate</td>
			<td>Fisher Scientific</td>
			<td>A13201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monoclonal anti-ceramide antibody (mouse IgM)</td>
			<td>Sigma</td>
			<td>C8104-50TST</td>
			<td>Clone: MID 15B4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat Anti-Mouse IgM Antibody, &#181; chain, FITC conjugate</td>
			<td>Sigma</td>
			<td>AP128F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fixation buffer</td>
			<td>Biolegend</td>
			<td>420801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Permeabilization buffer</td>
			<td>Ebioscience</td>
			<td>4307693</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Red Blood Cell Lysis Buffer&#160;</td>
			<td>Sigma</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Annexin V Binding Buffer</td>
			<td>BD Biosciences</td>
			<td>556454</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 cells</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning&#160;Cell Lifter&#160;</td>
			<td>Fisher Scientific</td>
			<td>07-200-364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:357px;height:21px;">Note: M.W. is for molecular weight.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

saturated fatty acids induce ceramide-associated macrophage cell death

Makrofager udtrykke meget epidermal fedtsyre-bindende protein og fedt fedtsyre-bindende protein. De aktivt optagelse mættede og umættede fedtsyrer, som kan spille en afgørende rolle i reguleringen af deres immun funktioner. Talrige undersøgelser har vist, at forskellige fedtsyrer, mættede eller umættede, kan have forskellige konsekvenser for cellevækst og funktion. Men tilgange bruges til fedtsyre forberedelse varierer, hvilket kan føre til ikke-fysiologiske resultater. Serum albumin, en naturlig transportør for fedtsyrer i pattedyr perifert blod, anbefales for at danne et kompleks med Natriumsalt af fedtsyrer konjugat at studere fedtsyre funktion i pattedyrceller, hvilket minimerer toksiciteten af fedtsyre sæbe. Således er en enkel, relativt hurtig opvarmning og sonicating metode udviklet og præsenteret her for BSA-fatty syre konjugat dannelse. Vi beskriver en protokol, ved hjælp af mættede fedtsyrer, især stearic syrer til at fremkalde alvorlige celledød i mus knoglemarv afledt makrofager. Vi viser yderligere, mættede fedtsyre-induceret celledød er positivt associeret med akkumulerede cellulære ceramid niveauer. Denne metode kan udvides til undersøgelser af virkningen af fedtsyre på andre pattedyrsceller.

Fedme øger frie fedtsyrer koncentrationer i serum. Som professionelle fagocytter tage makrofager aktivt op fedtsyrer til at opretholde vært homøostase. Under disse processer, kan overbelastet lipider fremkalde makrofag celledød. Med henblik herpå kulturperler vi BMDMs i vitro med fede niveauer af kosten fedtsyrer og målte makrofag celledød ved hjælp af flow cytometric farvning. I forhold til kontrolelementet BSA, mættede fedtsyrer, induceret navnlig stearic syrer, betyde...

Watch this Scientific Journal Video about Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death at JoVE.com

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Vi illustrere en ligetil metode til at udlede murine primære makrofager fra knoglemarven cellerne og en enkel metode til at forberede BSA-fedtsyre konjugater. Så vi demonstrere at mættede fedtsyrer kan fremkalde makrofag celledød, og sådanne celledød er positivt associeret med cellulære ophobning af ceramid niveauer.

Mættede fedtsyrer fremkalde ceramid-associerede makrofag celledød

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

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