Name: selective harvesting of marginating-hepatic leukocytes
Uploaded: 2016-07-21T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).

伦理学声明:涉及动物主题程序已在特拉维夫大学被批准的机构动物护理和使用委员会（IACUC）。 1.大鼠协议<ol><li>准备工作<ol><li>制备肝素的PBS（30单位/ ml）溶液在室温（RT）加入每毫升磷酸盐的不含防腐剂的肝素的30个单位被用来缓冲盐水（PBS）1×溶液。计算每个动物35毫升。 </li><li>通过蠕动泵线和蝴蝶针传递肝素的PBS，以消除气泡。 </li><li>消毒手术工具 - 2对剪刀，钝边镊子，2止血，和牙齿组织镊（高压釜在121℃下对重力（干燥）设定为至少30分钟）。 </li></ol></li><li> MH-白细胞的肝脏和收集灌注<ol><li> 8％的异氟醚过量安乐死的动物。作为预防措施，将含有异氟醚50ml管周围的老鼠头-soaked垫，直到其打开胸腔。 </li><li>打开腹腔和胸腔空腔后呼吸停止，立即露出肝脏和心肺复杂。避免通过这个过程出血。 <ol><li>具体而言，对角开始在下腹正中一点进步两侧切口，不破坏内脏，向排骨，通过它们切割胸上部腔水平。 </li></ol></li><li>在大约分钟，收集尽可能多的血越好（〜从250克动物6ml）中自右心室到注射器中。 </li><li>夹紧尾腔静脉尽可能接近到心脏止血钳，使灌流液的收集。 </li><li>拉出肠子和地点动物实验者的权利外，揭露门静脉。 </li><li>插入地下25 IV导管，连接到蠕动泵，进入门静脉，如尾越好，但喙脾静脉。 </li><li>插入地下25蝴蝶针，连接到5毫升注射器，进入下腔静脉，尾椎止血。 </li><li>打开蠕动泵以约3毫升/分钟的速度，并轻轻收集第一毫升被污染的血液到注射器灌注液。继续，直到灌流液颜色变成浅的红色（约3-5毫升）。注意，肝脏的颜色向浅棕色的变化。 </li><li>不停止蠕动泵，替换20ml的收割注射器中的5毫升收获注射器和收集至少20毫升采用灌注的更高的速度（最多4毫升/分钟）肝灌注液。连续监视灌注液流入收集注射器，并且避免真空是太强（这可通过折叠腔静脉壁堵塞针）。 </li><li>终止灌注时肝脏的颜色即变为浅棕色。 </li></ol></li><li>提取白细胞<o升&gt; <li>离心10分钟灌注液在400 xg离心24 C。 </li><li>吸出上清液</li><li>加入10毫升的PBS，离心，在400×g离心10分钟，吸出上清液。根据学习目标，用准备原样，或进行额外的纯化（ 如密度梯度分离）。 <ol><li>具体地，层4毫升灌洗液超过4毫升密度梯度分离质量，在50毫升聚丙烯离心管中。旋管在754 xg离心，RT，用于与所述脱离30分钟。 </li><li>获得在通过手动移液的密度梯度分离的上清液接口单核层，在PBS中洗涤，并插入到5ml的管中。 </li></ol></li></ol></li></ol> 2.鼠标协议<ol><li>准备工作<ol><li>通过每毫升磷酸盐加入不含防腐剂的肝素的30个单位制备肝素的PBS（30单位/ ml）溶液在室温（RT）的使用缓冲盐水（PBS）1×搜索解决方案ñ。计算每个动物20毫升</li><li>通过蠕动泵线和蝴蝶针传递肝素的PBS，以消除气泡。 </li><li>消毒手术工具 - 2对剪刀，钝边镊子，2止血，和牙齿组织镊（高压釜在121℃下对重力（干燥）设定为至少30分钟）。 </li></ol></li><li> MH-白细胞的肝脏和收集灌注<ol><li> 8％的异氟醚过量安乐死的动物。作为预防措施，将含有约鼠标头部异氟醚浸泡垫15毫升管，直至其打开胸腔。 </li><li>固定鼠标的四肢在床上。 </li><li>打开腹腔和胸腔空洞，露出肝脏，并立即心肺复时呼吸停止，保持完好的隔膜的高宽（以创建一个引流胸腔池）。避免通过这个过程出血。 <ol><li>具体而言，在较低的启动切口腹部中线来看，不损伤内脏，进步到两侧朝斜筋，通过它们切割胸上部腔水平。 </li></ol></li><li>拉出肠子和地点动物实验者的权利外，揭露门静脉。 </li><li>插入30克针，连接到蠕动泵向门静脉喙脾静脉。 </li><li>切隔膜上方的下腔静脉，以允许从胸腔灌注液引流和收集。 </li><li>打开蠕动泵以约3毫升/分钟的速度，并轻轻收集前面的3 mL被污染从胸腔池血液灌注液。 </li><li>弃掉此灌流液。如果需要的话再重复这样的洗涤，使用生理盐水，然后才开始收集肝脏灌注。 </li><li>重新发起灌注在多达4毫升/分钟的速度，并且使用从胸腔收集10毫升灌注液连接到注射器蝴蝶。 </li><li>终止灌注时肝脏的颜色即变为浅棕色。 </li></ol></li><li>提取白细胞<ol><li>离心机灌注液在400×g下10分钟。 </li><li>吸出上清液。 </li><li>加入10毫升的PBS，离心，在400×g离心10分钟，吸出基于学习目标上清液，按原样使用制备，或进行额外的纯化（ 例如 ，密度梯度分离）。 <ol><li>具体地，每4毫升灌洗液超过4毫升密度梯度分离质量，在50毫升聚丙烯离心管层。旋管在754 xg离心，RT，用于与所述脱离30分钟。 </li><li>获得在通过手动移液的密度梯度分离的上清液接口单核层，在PBS中洗涤，并插入到5ml的管中。 </li></ol></li></ol></li></ol>

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本文提出的肝脏灌注方法使选择性采伐和marginating肝白细胞的独特人口的研究。肝NK细胞，也被称为坑细胞3，构成驻留在肝窦鲜明的NK细胞群。它们在大鼠，小鼠17和在人类18,19找到。相比孤立外周血NK细胞，坑的细胞表现出较高的细胞毒性对YAC-1和CC531s靶细胞系20中 ，显示出具有更高的数目较小的胞质颗粒20，和表面抗原，诸如LFA-1的12的高表达。另?...

在肝血窦包含对生物体关键的各种免疫活动，众多的白细胞亚型。例如，marginating肝（MH +）自然杀伤（NK）细胞，也被称为坑的细胞，特征在于形态为大颗粒淋巴细胞（LGLs）和功能作为自发性细胞毒性的能力的白细胞，使肝-抵抗建立血 - 源性肿瘤转移。本文提出的方法的目标是使氢白细胞选择性收割，为了研究这个重要和独特的细胞群体（和免疫隔室），并阐明各种操作对这些特定细胞的影响（ 例如 ，免疫激活）。 尽管免疫的故障消除显影原发性肿瘤，在癌症患者和动物模型的证据表明免疫系统可控制循环肿瘤细胞，微转移，和残留病THROUGH细胞介导的​​免疫（CMI）。然而， 在体内的功能和在人的体外研究和在动物中，这表明，大多数的自体肿瘤细胞是通过循环或脾白细胞1,2-细胞毒性抗这些之间的明显不一致。此不一致可能归因，至少部分地明显的白细胞亚群，即marginating肝（正弦）白细胞和其活化NK细胞，即坑细胞3的亚群在体内存在。的确，从我们的实验室未发表的数据表明，在F344大鼠的同基因肿瘤细胞（MADB106），其被发现循环和脾白细胞抗性，通过MH-NK细胞4裂解。因此，是所谓的"NK抗性"，以循环白细胞的肿瘤细胞可以通过MH-NK细胞来控制。值得注意的是，在小鼠中只有下列体内免疫MH-NK的增强的活性是明显的刺激（ 例如，通过使用保利我的:C或CpG的-C）5。 肝特异性NK细胞（MH-NK）位于正弦管腔内，附着在血管内皮细胞和枯否氏细胞。 MH-NK细胞是专门特征在于球形致密颗粒和杆芯囊泡6，其含有酸性磷酸酶如溶酶体酶，和穿孔和粒酶作为生物活性物质7,8。与循环NK细胞相比，MH-NK细胞表现出的颗粒和囊泡9-11的较高数目和大小。下炎性病症，被示出的MH-NK细胞表现出的LFA-1 12的更高的表达相比，循环NK细胞。这种增强的表达可能构成了一个机制，MH-NK细胞是针对某些肿瘤细胞比循环NK细胞13,14多种细胞毒性。 nterestingly， 下列体外孵育白细胞介素（IL）-2，MH-NK细胞变大，并它们的数量和颗粒的尺寸增加，所有这些都与一个亲音响淋巴因子激活的杀伤的勒细胞（LAK）15相一致。 活性氢白细胞不能通过的标准肝白细胞采收方法，其基于研磨和组织的生物降解独占获得。相比于标准的方法本文描述的我们的灌注方法有两个主要优点。首先，灌注的方法选择性地收割MH白细胞，防止污染由其他肝病车厢其他白细胞。其次，灌注技术更好保持完整性，活性，和MH白细胞的生理环境，不像组织处理方法损害细胞或改变它们的形态，和由于组织损伤，诱导各种因素的释放是显着调节免疫活动。 肝脏是癌转移所涉及的主要靶器官第二对各种感染16。当MH细胞表现出独特的特点是，研究在各种条件下该特定人群相对于这些疾病是很重要的。例如，它是值得注意的各种生物反应调节剂是全身性免疫激活（ 如聚I:C或CpG的-C）已经显示激活比循环白细胞5 MH-白细胞多。

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26 G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21 G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>22 W Fluorescent Daylight Magnifier Lamp</td><td></td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-hepatic leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

在F344大鼠我们比较MH-NK细胞的以下肝组织的机械研磨的细胞毒性（从通过强制肝灌注的肝血窦收集）到整个肝脏细胞群的细胞毒作用，并以循​​环白细胞的细胞毒性。所有细胞制备物洗涤至少3次，如在免疫学​​测定程序中，并作为靶细胞系，我们使用了同种异体YAC-1或同基因MADB106靶细胞系。如在图1所示- 4，而MH-NK细胞表现出对MADB106和YAC-1靶?...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

selective-harvesting-of-marginating-hepatic-leukocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.6K 次观看.  Tel-Aviv University. Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

视频: Selective Harvesting of Marginating-hepatic Leukocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

发育生物学

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating肺白细胞的选择性收获

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

生物学

使用磁性细胞选择建立体外 LLEC 培养物

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

软脑膜淋巴内皮细胞的收获和原代培养

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

神经科学

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

医学

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Marginating肝白细胞的选择性收获

摘要

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此视频中的章节

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

分离和新型表面标记组合来标识肝祖亚群富集

Marginating肺白细胞的选择性收获

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

软脑膜淋巴内皮细胞的收获和原代培养

小鼠肝部分切除术后再生肝细胞的分离

小鼠原发性肝正弦内皮细胞的分离与表征