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胫骨/股骨骨的两端。将25克针与 PBS + 2% 胎牛血清 (FBS) 一起冲洗到15毫升管中. </li>
	<li> 在 500 x g、4和 #176 上离心骨髓细胞; C 为5分钟. </li>
	<li> 重1毫升红细胞裂解缓冲液中的细胞团溶解红细胞 (RBC) 少于1分钟, 立即用9毫升 1x PBS 稀释。再离心和在步1.3。醒酒清. </li>
	<li> 重10毫升 1x PBS 中的细胞颗粒, 通过不育的40和 #956 过滤细胞悬浮; #160; 尼龙网进入另一个15毫升管去除细胞碎片/团簇。再离心机作为在1.3。醒酒清. </li>
	<li> 重15毫升罗斯威尔公园纪念研究所中型 (RPMI) 1640 与 5% FBS 和10和 #956 的细胞颗粒; 庆大霉素, 将100毫米的组织培养皿中的细胞放在 37 o C 孵化箱中, 60 分钟. 轻轻地旋转盘子, 取出漂浮单元格, 并对它们进行计数. </li>
	<li> 板 6x10 6 12 毫升巨噬细胞分化培养基的100毫米盘中的细胞 (RPMI 1640 与 5% FBS 和10和 #956; 庆大霉素, 混合 30% L929 条件培养基 10 ) 与 10 ng/毫升重组小鼠巨噬细胞集落刺激因子 (M CSF) 为2天. </li>
	<li> 在37和 #176 中培养了2天后; C 孵化器与 5% CO 2 , 用6毫升新鲜巨噬细胞与 10 ng/毫升 M-CSF 喂养每道菜. </li>
	<li> 在5天, 采取8毫升的旧媒体, 而不干扰细胞和增加10毫升新鲜巨噬细胞分化介质与 10 ng/毫升 M-CSF. </li>
	<li> 在7天, 使用细胞提升器获取骨髓衍生巨噬细胞 (BMDMs). 
	注: 在 1x pbs 中, 附加的巨噬细胞也可与5毫升1毫米的 EDTA 分离, 5 至10分钟, 去除浮点数和洗涤板两次, 与5毫升 1x pbs。离心机的细胞, 在步骤 1.3., 重他们在新鲜巨噬细胞区分媒体, 并数他们与例。
	<ol> <li> 确认 BMDMs 和 #39; 表型由流式细胞仪描述为 5 。准备在一定浓度下的衍生小鼠巨噬细胞进行以下研究. </li>
	</ol> </li> </ol> 2。脂肪酸共轭制剂 <ol> <li> 在无菌 PBS 中制备2毫米 bsa。例如, 重6.65 克 bsa, 加入30毫升 1x pbs, 以解散, 然后添加更多的 pbs, 直到50毫升, 缩放组件, 使2毫米 bsa 在 1x pbs. </li>
	<li> 在2毫米 BSA 中制备5毫米的脂肪酸单独钠盐。加热到37和 #176; C 或更高, 如有必要, 几种2毫米 BSA 和脂肪酸钠的混合物, 直到得到明确的溶液为止. 
	注: 与不饱和脂肪酸相比, 饱和脂肪酸需要更高的温度和更超声的溶解时间. </li>
	<li> 通过0.22 和 #956 过滤脂肪酸 BSA 溶液, 将其分为1.5 毫升无菌离心管, 并将其存储在4和 #176; c 用于短期或-20 和 #176; c 用于长期存储。4、#176 贮存后不应出现降水. </li>
</ol> 3。饱和脂肪酸诱导小鼠骨髓源性巨噬细胞死亡的研究 <ol> <li> 在巨噬细胞分化介质中的24个组织培养板中 BMDMs 0.4 x 10 6 /毫升. </li>
	<li> 将 0.4 mM 棕榈酸和硬脂酸的细胞处理为16到24小时, 在37和 #176; C 孵化器与 5% CO 2 。使用 BSA 作为负控制. </li>
	<li> 在治疗后用细胞升降器采集细胞, 并在 500 x g、4和 #176 中旋转细胞; C 为5分钟. </li>
	<li> 在100和 #956 中用蛋白 v-Alexa 488 和 7-aminoactinomycin D (7-反倾销) 对细胞进行染色; 在室温下为15分钟蛋白 v 绑定缓冲区. </li>
	<li> 用200和 #181 稀释样品; 蛋白结合缓冲液, 轻轻搅拌, 在潜伏期后将样品保存在冰上. </li>
	<li> 通过流式细胞仪来尽快分析染色细胞 5 。在 FITC 通道中检测到蛋白 V-Alexa 488, 在 PerCP/PerCP-Cy5.5 通道中检测到 7-反倾销. </li>
</ol> 4。胞内神经酰胺染色 <ol> <li> 在脂肪酸处理后, 在步骤3.3 中收获细胞。然后将4% 甲醛中的单元格修复为 30 min. </li>
	<li> 用1毫升 1x PBS 清洗样品, 在3.3 中向下旋转一步, 醒酒上清液. </li>
	<li> 在100和 #956 中用 anti-ceramide 原抗体 (1:200 稀释) 将细胞染色; L 1x 性缓冲30分钟 </li>
	<li> 在步骤4.2 中再次清洗和旋转单元格. </li>
	<li> 在100和 #956 中用 anti-mouse IgM 二次抗体 (1:500 稀释) 将细胞染色; L 1x 性缓冲器30分钟 </li>
	<li> 在步骤4.2 中再次清洗和旋转单元格。添加250和 #956; 1x PBS 并通过流式细胞仪分析染色细胞 5 . 
	注: 请参阅设备和试剂表中的详细试剂清单. </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. 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作者声明没有利益的财政冲突。

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 ...

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

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Immunology and Infection

9.2K 次观看.  University of Louisville. 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。

视频: 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.

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 
insulin ...

科研

免疫与感染

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.

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

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.

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

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.

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 ...

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纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

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.

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

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.

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

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.

巨细胞动脉炎叶酸受体贝塔巨噬细胞和血管免疫微环境的免疫组织病理学分析

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.

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

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.

通过生命内显微镜在斑马鱼胚胎的分孔细菌感染过程中巨噬细胞细胞死亡的可视化

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