Diese Arbeit wurde teilweise unterstützt von der University of Louisville Startkapital und National Cancer Institute (Bethesda, MD) gewährt R01CA177679, R01CA180986.

das Protokoll von der institutionellen Animal Care und Nutzung Committee (IACUC) der University of Louisville gebilligt wurde.
 1. Maus Knochenmark abgeleitet Makrophagen (BMDMs) <ol><li>einschläfern eine 6 bis 8 Wochen alten, gesunden Wildtyp-Maus mit CO 2. Heften Sie es auf eine Dämmplatte und sprühen Sie es mit 70 % Ethanol, bis es getränkt ist. Entfernen Sie die Tibia/Femur-Knochen mit Zange und Schere. Setzen Sie sie in einer Petrischale mit 5 mL 1 x Phosphat-gepufferte Kochsalzlösung (PBS). Führen Sie alle Verfahren unter sterilen Bedingungen.</li>
	<li>Aufschneiden beide Enden der Tibia/Femur-Knochen mit einer Schere in einem sterilen Gewebekultur-Haube. Spülen Sie den Knochen mit einer 25 G Nadel mit PBS + 2 % fetalen bovine Serum (FBS) in einem 15 mL-Tube.</li>
	<li>Zentrifugieren Sie die Knochenmarkzellen 500 X g, 4 ° C für 5 min.</li>
	<li>Aufschwemmen der Zelle Pellet in 1 mL roten Blutzelle Lysis Puffer zur lyse der Erythrozyten (RBC) für weniger als 1 min. sofort mit 9 mL 1 X PBS verdünnt. Zentrifuge wieder wie in Schritt 1.3. Die Überstände dekantieren.</li>
	<li>Aufschwemmen der Zelle Pellet in 10 mL 1 X PBS, filtern die Zellsuspension durch eine sterile 40 μm-Nylon-Netzgewebe in einem anderen 15 mL-Tube, Zelle Schutt/Klumpen zu entfernen. Zentrifuge wieder wie in 1.3. Die Überstände dekantieren.</li>
	<li>Aufschwemmen der Zelle Pellet in 15 mL Roswell Park Memorial Institute mittlerer (RPMI) 1640 mit 5 % FBS und 10 μg/mL Gentamicin, Platte, die die Zellen in der Kulturschale von 100mm Gewebe am 37 o C-Inkubator für 60 min. Wirbel sanft das Gericht, die schwimmende herausnehmen Zellen, und zähle sie.</li>
	<li>Platte 6 x 10 6 Zellen in einer 100-mm-Schale mit 12 mL Makrophagen differenzieren Medien (RPMI 1640 mit 5 % FBS und 10 μg/mL Gentamicin, gemischt mit 30 % L929 konditionierten Media 10) mit 10 ng/mL rekombinanter Maus Makrophagen Kolonie-stimulierende Faktor (M-CSF) für 2 Tage.</li>
	<li>Nach Kultivierung für 2 Tage in einem Inkubator 37 ° C mit 5 % CO 2, füttern Sie jedes Gericht mit 6 mL frische Makrophagen differenzieren Medien mit 10 ng/mL M-CSF.</li>
	<li>Am Tag 5, 8 mL der alten Medien, ohne die Zellen zu stören und fügen 10 mL frisches Makrophagen differenzieren Medien mit 10 ng/mL M-CSF.</li>
	<li>Am 7. Tag ernten die Knochenmark stammenden Makrophagen (BMDMs) mit einer Zelle Heber. 
	Hinweis: Angehängte Makrophagen können auch mit 5 mL 1 mM EDTA mit 1 X PBS-Puffer für 5 bis 10 min losgelöst werden nach dem Entfernen der Float-Zellen und Platte zweimal mit 5 mL 1 X PBS waschen. Zentrifugieren Sie die Zellen wie in Schritt 1.3., Aufschwemmen sie in frische Makrophagen, die Medien zu unterscheiden und mit einer Hemocytometer zu zählen.
	<ol><li>Bestätigen die BMDMs &#39; Phänotyp durch Durchflusszytometrie als 5 beschrieben. Vorbereiten der abgeleiteten Maus Makrophagen in einer bestimmten Konzentration für die folgenden Studien.</li>
	</ol></li></ol> 2. BSA-fetthaltige Säure konjugieren Vorbereitung <ol><li>vorbereiten 2 mM BSA in sterilen PBS. Zum Beispiel wiegen 6,65 g BSA, hinzufügen 30 mL 1 X PBS aufzulösen, fügen Sie mehr PBS bis 50 mL Skalierung Komponenten um 2 mM BSA in 1 X PBS zu machen.</li>
	<li>Vorbereitung 5 mM individuelle Natriumsalz der Fettsäuren in 2 mM BSA. Heizen auf 37 ° C oder höher, wenn nötig, und beschallen die Mischung von 2 mM BSA und Natriumsalz von Fettsäuren, bis eine klare Lösung erhalten wird. 
	Hinweis: Im Vergleich zu ungesättigten Fettsäuren, gesättigter Fettsäuren benötigen eine höhere Temperatur und weitere Beschallungsdauer für auflösen.</li>
	<li>Filter die Fettsäuren-Säure-BSA-Lösung durch einen 0,22-μm-Filter, aliquoten es in 1,5 mL sterile Microcentrifuge Rohre, und speichern sie bei 4 ° C für den kurzfristigen Einsatz oder bei-20 ° C für langfristige Lagerung. Kein Niederschlag nach Lagerung bei 4 ° C im Kühlschrank beachtet werden.</li>
</ol> 3. Gesättigte Fettsäuren induzieren Zelle Tod der Maus Knochenmark abgeleitet Makrophagen <ol><li>BMDMs in einer Gewebekultur 24-Well-Platte-Platte mit 0,4 x 10 6 /mL in Makrophagen differenzieren Medien.</li>
	<li>Behandlung der Zellen mit 0,4 mM Palmitinsäure und Stearinsäure für 16 bis 24 h bei 37 ° C-Inkubator mit 5 % CO 2. BSA zu verwenden, als die Negativkontrolle.</li>
	<li>Ernte der Zellen mit einer Zelle Lifter nach Behandlung und spin-down der Zellen bei 500 X g, 4 ° C für 5 min.</li>
	<li>Beflecken Zellen mit Annexin V-Alexa 488 und 7-Aminoactinomycin D (7-AAD) in 100 μL annexin V Bindung Puffer für 15 min bei Raumtemperatur.</li>
	<li>Die Probe mit 200 µL annexin-Bindung Puffer verdünnen, vorsichtig mischen, dann halten Sie die Proben auf Eis nach der Inkubationszeit.</li>
	<li>Analysieren die gefärbten Zellen so bald wie möglich durch Flow Cytometry 5. Annexin V-Alexa 488 in FITC Kanal erkannt und 7-AAD in PerCP/PerCP-Cy5.5-Kanal erkannt wird.</li>
</ol> 4. Intrazelluläre Ceramid Färbung <ol><li>nach der Fettsäure-Behandlung, die Zellen wie in Schritt 3.3 ernten. Befestigen Sie die Zellen in 4 % Paraformaldehyd für 30 min.</li>
	<li>Die Probe mit 1 mL 1 X PBS waschen, spin-down als Schritt in 3.3 und dekantieren des Überstandes.</li>
	<li>Die Zellen mit Anti-Ceramid Primärantikörper (Verdünnung 1: 200) in 100 μl 1 X Permeabilisierung Puffer für 30 min. Färben</li>
	<li>Waschen und Spin down die Zellen wieder wie in Schritt 4.2.</li>
	<li>Die Zellen mit Anti-Maus IgM Sekundärantikörper (Verdünnung 1: 500) in 100 μl 1 X Permeabilisierung Puffer für 30 min. Färben</li>
	<li>Waschen und Spin down die Zellen wieder wie in Schritt 4.2. Fügen Sie 250 μl 1 X PBS und analysieren die gefärbten Zellen von Flow Cytometry 5. 
	Hinweis: Finden Sie die detaillierte Reagenz-Liste in der Tabelle von Geräten und Reagenzien.</li>
</ol>

<ol>
	<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. E., Casiano, C. A., De Leon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+and+stearic+fatty+acids+induce+caspase-dependent+and+-independent+cell+death+in+nerve+growth+factor+differentiated+PC12+cells.">Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells.</a> J Neurochem. 84 (4), 655-668 (2003).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. , (2008).</li><li>Dansen, T. B., 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=High-affinity+binding+of+very-long-chain+fatty+acyl-CoA+esters+to+the+peroxisomal+non-specific+lipid-transfer+protein+(sterol+carrier+protein-2).">High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).</a> Biochem J. 339 (Pt 1), 193-199 (1999).</li><li>Ulloth, J. E., 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=Characterization+of+methyl-beta-cyclodextrin+toxicity+in+NGF-differentiated+PC12+cell+death.">Characterization of methyl-beta-cyclodextrin toxicity in NGF-differentiated PC12 cell death.</a> Neurotoxicology. 28 (3), 613-621 (2007).</li></ol>

Die Autoren erklären keine finanziellen Interessenkonflikte.

Die richtige Vorbereitung der Fettsäure-Lösung ist von entscheidender Bedeutung für die biologische Funktion von Fettsäuren zu studieren. Die Neutralisation der Fettsäuren erhöht die Löslichkeit in wässriger Lösung. Natriumsalze der Fettsäuren, vor allem gesättigte Fettsäuren, sind jedoch noch der geringe Löslichkeit in Wasser oder PBS, wie wir beobachten. Eine Methode ist mit 95-100 % Ethanol helfen Fettsäure1auflösen. Mit dieser Methode kann auch bei niedrigeren Konzentrationen f?...

Fettsäuren spielen eine wichtige Rolle im Energiestoffwechsel und in der Synthese der Phospholipide der Membran in verschiedenen Arten von Zellen. Fettsäuren haben eine geringe wässrige Löslichkeit. Entsprechende Vorbereitung der Fettsäure ist von entscheidender Bedeutung für die Erforschung der biologischen Funktionen der Fettsäuren in Säugerzellen. Wenn Fettsäuren mit Ethanol vorbereitet sind, kann viele Fettsäuren ihre giftigen Seife (Reinigungsmittel) Wirkung auf die Zellmembran, auch bei relativ niedrigen Konzentrationen1angezeigt. Als natürliche, große Transporter für die freien Fettsäuren im Serum gilt als Serum-Albumin einen guten Träger für Fettsäure-Lieferung in Vitro für Fettsäure Funktion Assays2,3,4. Jedoch sind die Details der Vorbereitung der Fettsäure und Serum-Albumin-Konjugat in der Regel nicht verfügbar, obwohl viele Forschungsarbeiten mit Fettsäuren veröffentlicht worden sind.
Makrophagen express hoch epidermale Fettsäure-bindendes Protein und Fett Fettsäure-bindende Protein5,6,7,8. Sie aktiv Aufnahme gesättigten und ungesättigten Fettsäuren, die ihre Immunfunktionen regulieren kann. Um die Wirkung von Fettsäuren auf Makrophagen und andere Zellen zu untersuchen, wurden verschiedene Methoden der Fettsäure Vorbereitung angewandte1,7,9. Mit entsprechend vorbereitete Fettsäure/Serum-Albumin-Konjugate, um die Wirkung von Fettsäuren auf Makrophagen Funktion zu untersuchen, ist von entscheidender Bedeutung in biologisch sinnvolle Daten zu erhalten. Studien über die Auswirkungen der Fettsäuren auf Makrophagen Funktion bieten Grundkenntnisse und mögliche therapeutische Ziele in Bezug auf Fettsäure-Stoffwechsel bei Erkrankungen Makrophagen beteiligt.

<table width="828">
	<tbody>
		<tr height="20">
			<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

Makrophagen express hoch epidermale Fettsäure-bindendes Protein und Fett Fettsäure-bindendes Protein. Sie aktiv Aufnahme gesättigten und ungesättigten Fettsäuren, die eine entscheidende Rolle bei der Regulierung der Immunfunktionen spielen könnte. Zahlreiche Studien haben gezeigt, dass verschiedene Fettsäuren, gesättigt oder ungesättigt, können unterschiedliche Auswirkungen auf das Zellwachstum und die Funktion besitzen. Allerdings variieren die Ansätze für die Fettsäure-Vorbereitung zu unphysiologischen Ergebnissen führen kann. Serum-Albumin, natürliche Träger für Fettsäuren in peripheren Säugetierblut wird empfohlen zur Bildung einer konjugiert komplex mit das Natriumsalz von Fettsäuren, Fettsäure-Funktion in Säugerzellen, minimiert die Toxizität von Fettsäure-Seife zu untersuchen. Somit ist eine einfache, relativ schnelle Heizung und beschallen Methode entwickelt und präsentiert hier für BSA-Fettsäuren konjugierte Säurebildung. Wir beschreiben ein Protokoll mit gesättigten Fettsäuren, vor allem Stearinsäure Säuren zu schweren Zelltod in der Maus Knochenmark abgeleitet Makrophagen induzieren. Wir zeigen weiter, dass gesättigte Fettsäure-induzierte Zelltod mit kumulierten zellulären Ceramid-Spiegel positiv assoziiert ist. Diese Methode kann für Studien über die Auswirkungen der Fettsäure auf andere Säugetier-Zellen erweitert werden.

Fettleibigkeit erhöht freie Fettsäure-Konzentrationen im Serum. Als professionelle Phagozyten aufgreifen Makrophagen aktiv Fettsäuren zu Host Homöostase. Bei diesen Prozessen können überladene Lipide Makrophagen Zelltod auslösen. Zu diesem Zweck wir BMDMs kultiviert in Vitro mit übergewichtigen diätetischen Fettsäuren und gemessenen Makrophagen Zelltod mit Flow durchflusszytometrischen Färbung. Im Vergleich zur BSA-Kontrolle, gesättigten Fettsäuren, induziert insbeso...

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

Wir zeigen eine geradlinige Methode murine primären Makrophagen von Knochenmarkzellen zur Ableitung und eine einfache Methode zur Vorbereitung von BSA-Fettsäure konjugiert. Dann zeigen wir, dass gesättigter Fettsäuren Makrophagen Zelltod induzieren können, und solchen Zelltod positiv assoziiert mit zellulären Akkumulation von Ceramid-Ebenen ist.

Gesättigte Fettsäuren induzieren Ceramid-assoziierten Makrophagen Zelltod

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

saturated-fatty-acids-induce-ceramide-associated-macrophage-cell-death

Research

JoVE Journal

Immunology and Infection

9.2K Aufrufe.  University of Louisville. Wir zeigen eine geradlinige Methode murine primären Makrophagen von Knochenmarkzellen zur Ableitung und eine einfache Methode zur Vorbereitung von BSA-Fettsäure konjugiert. Dann zeigen wir, dass gesättigter Fettsäuren Makrophagen Zelltod induzieren können, und solchen Zelltod positiv assoziiert mit zellulären Akkumulation von Ceramid-Ebenen ist.

Serie: Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Methoden zur Beta Cell Death vermittelt durch zytotoxische T-Lymphozyten Beurteilung

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Forschung

Immunologie und Infektion

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Untersuchung von Makrophagen-Polarisation mit Knochenmark-Makrophagen

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Bestimmung der tolerierbaren Fettsäuren und Cholera Toxin-Konzentrationen mit menschlichen Darm-Epithelzellen und BALB / c Maus-Makrophagen

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Knochenmark-abgeleiteten Makrophagen-Produktion

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA-Reinigung aus intrazellulär Grown<em&gt; Listeria monocytogenes</em&gt; In Makrophagenzellen

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Visualisierung von Makrophagen extrazelluläre fallen mit der konfokalen Mikroskopie

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Erkennung von Inflammasome Aktivierung und Pyroptotic Zelltod in murinen Makrophagen Knochenmark abgeleitet

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Eine Immunohistopathologic Untersuchung der Folat-Rezeptor Beta Makrophagen und vaskuläre immun Mikroumgebung in riesige Zelle Arterienentzündung Profil

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Verarbeitung von Bronchoalveolar Lavage Fluid und Matched Blood für Alveolar Macrophage und CD4+ T-Zell Immunophenotypisierung und HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Visualisierung des Makrophagen-Lytic Zelltodes während der mykobakteriellen Infektion bei Zebrafischembryonen mittels Intravital-Mikroskopie

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...