This work was funded by a research grant and a Grant-in-Aid from the Food Nanotechnology Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.

1. Perfusion of the Liver 
<ol>
 <li>Anesthetize an adult male Sprague-Dawley rat (at 10 to 15 weeks-old; body weight 150-200 g) by an intraperitoneal injection of a sodium pentobarbital solution (50 mg sodium pentobarbital / kg body weight).</li>
 <li>Place the animal in a stainless pan and spray 70% ethanol onto its abdomen and thorax.</li>
 <li>Make a small cut and peel off the skin. Open the abdomen by the cut with a pair of scissors.</li>
 <li>Confirm the location of portal vein, pass a surgical thread under the portal vein and loosely clump.</li>
 <li>Clump the distal part of the inferior vena cava and mesenteric vein to prevent blood reflux into the liver. Open the thoracic cavity by cutting the diaphragm with scissors, and expose the heart.</li>
 <li>Insert a plastic catheter (2 mm diameter) into the portal vein and tighten the thread firmly around the catheter.</li>
 <li>Connect the catheter to a perfusion system which is loaded with washing solution (Ca2+-Mg2+free HBSS containing 0.5 mM EGTA, 10 mM HEPES and 4.2 mM NaHCO3 ; pH 7.2) prewarmed at 37 &deg;C.</li>
 <li>Begin perfusion in situ at a rate of 10 ml/min. At the same time, make a cut in right atrium wall. Continue perfusion for 10 min.</li>
 <li>Switch the perfusion solution to collagenase solution [HBSS containing 10mM HEPES and 4.2mM NaHCO3 supplemented with Type I collagenase (0.05%) and trypsin inhibitor (50 &mu;g/ml); pH 7.5], and perfuse for 10 - 20 min at a rate of 10 ml/min until the liver slightly swells and shows the sign for disintegration of the liver structure under serous membrane.</li>
</ol>
2. Preparation of Parenchymal Hepatocyte-rich Cell Fraction
<ol>
 <li>Remove and carefully transfer the liver into a sterile beaker containing 25 ml of collagenase solution. Mince into small pieces by scissors.</li>
 <li>Add 75 ml of cold MEM into the resultant cell suspension. After gentle pipetting, filtrate through a cell strainer (100 &mu;m)to remove connective tissues and undigested tissue fragments.</li>
 <li>Transfer the filtrate into 50 ml conical tubes and centrifuge at 50 x g for 1 min at 4&deg;C (brake off).</li>
 <li>Carefully discard the supernatant. Resuspend the pellet with cold MEM.</li>
 <li>Repeat 2.4) three times.</li>
</ol>
3. Mixed Primary Culture of Liver Cells 
<ol>
 <li>Suspend the cells obtained in 2.5) in the growth medium composed of DMEM containing 10% heat inactivated FCS, supplemented with 100 &mu;M &#946;-mercaptoethanol, 10 &mu;g/ml insulin, 100 &mu;g/ml streptomycin and 100 U/ml penicillin, and seed into five to ten tissue culture flasks (surface area: 75 cm2) at a density of 6.7 x 104 cells / cm2 (5 x 106 cells / flask / 12 -15 ml of medium).</li>
 <li>Incubate the culture flasksat 37&deg;C in an atmosphere of 5% CO2- 95% air.</li>
 <li>Replace the growth medium every 2 to 3 days intervals.</li>
</ol>
4. Selective Isolation of Macrophages
<ol>
 <li>After 12 days of culture, macrophages proliferate on the cell sheet. These cells are suspended by reciprocal shaking of the culture flasks at 80 - 120 strokes per minute for 30 min at 37 &deg;C.</li>
 <li>Transfer the culture medium into 100 mm non-tissue culture grade plastic dishes. Pool the medium from two T75 flasks into one plastic dish. Refill culture flasks with the growth medium and return them to the incubator.</li>
 <li>Incubate plastic dishes for 30 min at 37 &deg;C in an atmosphere of 5% CO2- 95% air. Macrophages readily attach to non-tissue culture grade plastic dishes under this incubation process, whereas other contaminating cells do not.</li>
 <li>Aspirate the medium and gently rinse the plastic dish with PBS. Repeat this step three times.</li>
 <li>Add 1 ml of TrypLE Express solution into the dish, and incubate for 10min at 37 &deg;C in an atmosphere of 5% CO2- 95% air.</li>
 <li>Add 9 ml of the growth medium. Gently scrape off the attached macrophages by a cell scraper. </li>
 <li>Transfer the cell suspension into a 15 ml conical tube and centrifuge at 180 x g for 5 min at 25&deg;C (brake on).</li>
 <li>Discard the supernatant and add 1 ml of the growth medium. Dissociate into single cells by vigorous pipetting. Enumerate the cell number by a hemocytometer-counting.</li>
 <li>Isolation steps can be repeated with the same culture flasks at two to three day intervals for more than two weeks.</li>
 <li>Isolated macrophages can be cultured in the growth medium described in 3.1.</li>
</ol>
5. Representative Results
An example of a mixed primary culture of adult rat liver cells is shown in Figure 2. Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells. Then, around day 6 to 12, phase contrast-bright, round macrophage-like cells vigourouly proliferate on the fibroblastic cell sheet. By shaking of the culture flasks, macrophages are readily suspended into the culture medium, and subsequent transfer and incubation in plastic dishes result in selective adhesion of macrophages(Figure 3). The macrophage-like cells can be harvested as early as day 8, and the numbers reached maximal levels on days 12 to 14. More than 106 cells can be harvested from a T75 culture flask repeatedly at 2 to 3 day intervals for more than two weeks, enabling a total cell yield per flask of 107 per T75 culture flask (Figure 3). The purities of the isolated macrophages were 95 to 99%, as evaluated by flowcytometry or immunocytochemistry with rat macrophage-specific antibodies,such as ED-1 (Figure 4), ED-3, OX-41 (Figure 4) and Iba19.The isolated cells show typical characteristics of macrophages, such as 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. 


<img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Figure 1" /> 
Figure 1. A scheme for selective isolation and purification of macrophage-like cells from mixed primary culture of rat liver cells.

<img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Figure 2" /> 
Figure 2. Primary culture of rat liver cells and proliferation of macrophage-like cells. Arrow indicates a multinuclear giant cell. Scale bar = 100 &mu;m. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010) 9 with permission from Elsevier.

<img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Figure 3" /> 
Figure 3. Selective isolation of macrophage-like cells by the shaking and attachment method. Cells were suspended into the culture medium by shaking the flasks, subsequently transferred into non-tissue culture grade plastic dishes, and incubated at 37 &deg;C. As early as 10 min after plating, macrophage-like cells attached to the dish surface, while other contaminating fibroblastic cells remained suspended. After rinsing with PBS, a highly purified macrophage population was obtained. These cells gained typical macrophage morphology after 40 min of culture, and mitotic cells were frequently observed (arrows). Subsequent changes in cell numbers recovered from flasks at different culture periods are shown. Values are an average&plusmn;SD from three to five flasks. Experiments were repeated three times and data are indicated by different symbols. Scale bar =100 &mu;m. Reprinted from the Journal of Immunological Methods, Vol.360, 47-55 (2010)9 with permission from Elsevier.

<img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Figure 4" /> 
Figure 4. Immunocytochemical staining of macrophage-like cells with monoclonal antibodies against rat macrophages.

<img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Figure 5" /> 
Figure 5. Phagocytosis of FITC-labeled microbeads by macrophage-like cells.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

All animals were kept and operated in the animal facility in the National Institute of Animal Health, according to the guidelines and regulations set forth by the Institution's Committee for animal experiments.

Here, we report a simple and efficient method to obtain macrophages from the mixed primary cultures of adult rat liver cells. Our method relies first on the novel proliferative activity of macrophages in the mixed culture of rat liver cells, and then subsequent isolation and purification of these cells on the basis of their biological characteristics as macrophages9. It may be possible the macrophages proliferated in the mixed primary culture of adult liver cells might have originated from Kupffer or related cells that contaminated into the parenchymal hepatocytes fraction. The macrophages might have responded to specific environmental changes caused by transformation of parenchymal hepatocyte10 and other fibroblastic cells in the mixed primary cultures.
In conclusion, our method provides isolation of macrophage-like cells in sufficient number and purity from the primary culture of rat liver cellswithout using sophisticated equipment and technical skills. In addition, the isolation procedure can be repeated with the same culture flask for more than two weeks. This method might be applicable to other mammalian species.

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Sodium pentobarbital solution</td>
 <td>Kyoritsu Seiyaku</td>
 <td>Somnopentyl Injection</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank's balanced salt solution (HBSS)</td>
 <td>Invitrogen</td>
 <td>14185-052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethylene glycol tetraacetic acid(EGTA)</td>
 <td>Sigma-Aldrich</td>
 <td>E-4378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Wako Pure Chemical Industries, Ltd.</td>
 <td>032-10534</td>
 <td>Cell dissociation grade 
 (Lot check needed)</td>
 </tr>
 <tr>
 <td>Trypsin inhibitor</td>
 <td>Sigma-Aldrich</td>
 <td>T-9128</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Eagle's minimum essential medium (MEM)</td>
 <td>Sigma-Aldrich</td>
 <td>M-4655</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's modified 
 Eagle's medium(DMEM)</td>
 <td>Sigma-Aldrich</td>
 <td>D-6459</td>
 <td>High glucose-type</td>
 </tr>
 <tr>
 <td>Fetal calf serum
 (FCS)</td>
 <td>HyClone</td>
 <td>SH30070.03</td>
 <td>Heat inactivated at 56&deg;C for 30 min. 
 (Lot check needed)</td>
 </tr>
 <tr>
 <td>TrypLE Express</td>
 <td>Invitrogen</td>
 <td>12605</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's phosphate buffered saline (PBS)</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td>Ca2+-Mg2+ free</td>
 </tr>
 <tr>
 <td>&#946;-mercaptoethanol</td>
 <td>Sigma-Aldrich</td>
 <td>M-7522</td>
 <td>Stock: 100 mM in distilled water</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I-5500</td>
 <td>Stock: 10 mg/ml in 0.1N HCl</td>
 </tr>
 <tr>
 <td>Penicillin/ streptomycin</td>
 <td>Invitrogen</td>
 <td>15070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell strainer</td>
 <td>BD Biosciences</td>
 <td>352360</td>
 <td>Mesh size: 100 &mu;m</td>
 </tr>
 <tr>
 <td>Tissue culture flask</td>
 <td>Sumitomo Bakelite Co., Ltd.</td>
 <td>MS-21250</td>
 <td>Surface area: 75 cm2</td>
 </tr>
 <tr>
 <td>Non-tissue culture plastic dishes</td>
 <td>BD Biosciences</td>
 <td>351005</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell scraper</td>
 <td>Corning Inc.</td>
 <td>3010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Reciprocal shaker</td>
 <td>TAITEC</td>
 <td>NR-1</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

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.

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

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38.1K 次观看.  National Institute of Agrobiological Sciences, Tsukuba, Japan. 获得原代培养大鼠肝细胞的巨噬细胞的一个新的方法来描述。这种方法利用在文化的巨噬细胞增殖，选择性附件晃动培养瓶和塑料菜的净化。这项技术有效地提供肝巨噬细胞，而无需复杂的设备和技能。

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...