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

这项工作由路易斯维尔大学启动基金和国立癌症研究所 (R01CA177679, R01CA180986) 资助。

该议定书得到了路易斯维尔大学机构动物保育和使用委员会 (IACUC) 的批准. 
 1. 小鼠骨髓衍生巨噬细胞 (BMDMs) <ol> <li> 安乐 6-至8周, 健康的野生型鼠标使用 CO 2 。把它钉在泡沫板上, 用70% 乙醇喷洒, 直到它被浸湿。用钳子和剪刀取出胫骨/股骨骨。用5毫升1x 磷酸缓冲盐水 (PBS) 将它们放入培养皿中。在无菌条件下执行所有程序. </li>
	<li> 在无菌组织培养罩中用剪刀切开?...

<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>

正确制备脂肪酸溶液对研究脂肪酸的生物学功能具有重要意义。脂肪酸的中和性增加了其在水溶液中的溶解度。然而, 我们观察到, 脂肪酸的钠盐, 特别是饱和脂肪酸, 在水或 PBS 中的溶解度仍然很低。一种方法是使用 95-100% 乙醇来帮助溶解脂肪酸1。使用这种方法, 即使在较低浓度的低毒性不饱和脂肪酸, 对细胞的毒性也会更高。另一种制备脂肪酸溶液的方法是利用甲基β-环糊精作?...

脂肪酸在能量代谢和不同类型细胞的膜磷脂合成中起着至关重要的作用。脂肪酸具有低水溶性。适当制备脂肪酸对研究哺乳动物细胞中脂肪酸的生物学功能具有重要意义。当脂肪酸准备用乙醇, 许多脂肪酸可能显示其有毒肥皂 (洗涤剂) 对细胞膜的影响, 即使在相对较低的浓度1。作为一个天然的, 主要运输者为游离脂肪酸在血清中, 血清白蛋白被认为是一个很好的载体脂肪酸传递在体外的脂肪酸功能检测2,3,4。然而, 尽管许多使用脂肪酸的研究论文已经出版, 但关于脂肪酸和血清白蛋白共轭物的制备的细节通常是不可用的。
巨噬细胞高表达表皮脂肪酸结合蛋白和脂肪脂肪酸结合蛋白5,6,7,8。他们积极摄取饱和和不饱和脂肪酸, 这可能调节他们的免疫功能。为了研究脂肪酸对巨噬细胞和其他组织的影响, 采用了不同的脂肪酸制备方法1,7,9。使用适当制备的脂肪酸/血清白蛋白结合物来研究脂肪酸对巨噬细胞功能的影响, 对于获得具有生物学意义的数据至关重要。研究脂肪酸对巨噬细胞功能的影响, 可为巨噬细胞相关疾病的脂肪酸代谢提供基础知识和潜在的治疗靶点。

<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

巨噬细胞高表达表皮脂肪酸结合蛋白和脂肪脂肪酸结合蛋白。他们积极摄取饱和和不饱和脂肪酸, 这可能在调节其免疫功能方面起到关键作用。许多研究表明, 各种脂肪酸, 饱和或不饱和, 可能对细胞生长和功能有不同的影响。然而, 用于脂肪酸制备的方法各不相同, 这可能导致 non-physiological 的结果。血清白蛋白是哺乳动物外周血中脂肪酸的天然载体, 推荐用脂肪酸钠盐组成共轭络合物, 研究哺乳动物细胞中的脂肪酸功能, 从而最大限度地减少脂肪酸皂的毒性。因此, 本文提出了一种简单、相对快速的加热和 sonicating 方法, 用于 BSA-脂肪酸共轭形成。我们描述了一个协议使用饱和脂肪酸, 特别是硬脂酸, 以诱导在小鼠骨髓衍生巨噬细胞死亡严重。我们进一步证明饱和脂肪酸诱导的细胞死亡与积累的细胞神经酰胺水平呈正相关。这种方法可以扩展到研究脂肪酸对其他哺乳动物细胞的影响。

肥胖会增加血清中游离脂肪酸的浓度。作为专业的吞噬, 巨噬细胞积极地服用脂肪酸来维持宿主的稳态。在这些过程中, 超负荷的脂质可能导致巨噬细胞死亡。为此, 我们培养 BMDMs体外与肥胖水平的膳食脂肪酸和测量巨噬细胞死亡使用流细胞染色。与 BSA 控制相比, 饱和脂肪酸, 特别是硬脂酸, 诱导 BMDMs 细胞死亡. 死细胞显示为双阳性种群染色的蛋白 V 和 7-反倾销 (<stron...

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

本文阐述了一种从骨髓细胞中提取小鼠原发巨噬蛋白的方法, 并提出了制备 BSA-脂肪酸共轭物的简便方法。然后我们证明饱和脂肪酸能诱导巨噬细胞死亡, 这种细胞死亡与神经酰胺水平的细胞积累呈正相关。

饱和脂肪酸诱导神经酰胺相关巨噬细胞死亡

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

Research

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Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

使用骨髓源性巨噬细胞巨噬细胞极化的调查

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

使用人类肠道上皮细胞和BALB / C小鼠巨噬细胞的容忍脂肪酸和霍乱毒素浓度的测定

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

骨髓衍生的巨噬细胞产

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

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

利用共焦显微镜可视化巨噬细胞胞外陷阱

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

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

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

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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

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

用于阿尔韦拉尔巨噬细胞和CD4和T细胞免疫造血和HIV储液评估的支气管性洗浴液和匹配血液的处理

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

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