The authors would like to thank Sally Roberts (University of Birmingham, Birmingham UK) for the kind gift of the E1&circ;E4 antibody. This work was supported by a grant from the National Cancer Institute (4R00CA137160-03).

1. Preparation for Organotypic Raft Cultures
<ol>
	<li>The metal raft grids much first be treated with chromic sulfuric acid to remove any residue that could interfere with the differentiation process. Immerse metal grids in a glass beaker containing sulfuric acid for one hour, then continuously rinsed overnight with tap water. After the overnight rinse, raft grids should be rinsed for 3-5 hours in double distilled water.</li>
	<li>To provide support for the rafts, bend three sides of the raft about 0.5 cm at equal distance from each other. The metal grids should then be autoclaved.</li>
	<li>Prepare the 10X reconstitution buffer by adding 2.2 g NaHCO3 and 4.8 g Hepes. Dissolve in 100 ml of 0.05 M NaOH. Filter sterilize and store in 5 ml aliquots at -20&deg;C.</li>
	<li>Prepare the 10X DMEM without sodium bicarbonate. Filter sterilize and store in 5 ml aliquots at -20&deg;C.</li>
	<li>Prepare mouse epidermal growth factor: Dissolve 100 mg of EGF and 10 mg of BSA each in 10 ml deionized ddH20. Combine EGF and BSA and add to 80 ml of deionized ddH20 for a total volume of 100 ml. Filter-sterilize, aliquot (5 ml) and store in the -20&deg;C. For each liter of E medium add 5 ml of 1 mg/ml EGF (final concentration 5 ng/ml).</li>
</ol>
2. Preparation of Collagen Gels
<ol>
	<li>One collagen gel is required for each raft. The collagen should be kept on ice to prevent it from solidifying. Each collagen gel requires 3 ml of collagen mix consisting of: 2.4 ml of cold Rat-tail collagen type I (final concentration 3-4 mg/ml), 0.3 ml 10X reconstitution buffer, 0.3 ml 10X DMEM and1-2 X 106 mouse 3T3 J2 fibroblasts as feeder cells. For high concentration solutions of rat tail collagen, dilute in 0.02 M acetic acid to achieve the correct working concentration.</li>
	<li>Determine the number of collagen gels (rafts) you will need and then calculate a master mix for the amount of collagen, 10X reconstitution buffer, 10X DMEM and J2 fibroblasts required. Include enough in each master mix for 3-4 extra gels due to the viscosity of the collagen.</li>
	<li>To prepare the collagen gel, trypsinize the J2 fibroblasts and neutralize with medium. Combine all fibroblasts and place in a 50 ml conical vial. Count the cells to determine the number of rafts that can be made. Again, 1-2x106 fibroblasts are required per collagen gel and thus per raft. Spin down the cells at low speed.</li>
	<li>Aspirate the medium from the fibroblast pellet and resuspend in the appropriate amount of 10X reconstitution buffer, 10X DMEM and collagen for the number of rafts calculated. Add the collagen last to prevent the gel from solidifying too quickly. Once the collagen is added, mix quickly, but gently to prevent the introduction of bubbles into the gel. The gel should be a reddish orange color, indicative of the correct pH. If the gel mixture is too yellow, add a couple of drops of filter-sterilized 1N NaOH and mix gently to obtain the correct color.</li>
	<li>Add 3 ml of the collagen:fibroblast mixture to the wells of a 6 well culture dish. The collagen mixture should be pipetted slowly down the side of each well to avoid generating bubbles. Place the plate in a 37&deg;C tissue culture incubator and allow to solidify for 30 minutes.</li>
	<li>After 30 minutes, add 3 ml of E medium with EGF to the top of the solidified collagen gel and return the plate to the incubator. The gels should be kept at 37&deg;C for at least one day but used within four days. Optimal results occur after two days of incubation.</li>
</ol>
3. Preparing Keratinocytes for Differentiation
<ol>
	<li>Low passage keratinocytes should be grown to approximately 70% confluency. Each raft requires 1-2x106 keratinocytes. Trypsinize the keratinocytes and neutralize with E medium with EGF. Count the cells to determine the number of rafts that can be made. Spin down the cells at low speed. Resuspend the pellet in 1 ml of E medium with EGF for each raft culture calculated.</li>
	<li>Aspirate the medium from the collagen gels by tilting the 6 well plate. Add 2 ml of fresh E medium with EGF to each gel. For each gel, carefully add 1 ml of resuspended keratinocytes to the 2 ml of E medium already present in the well. Gently pipet the keratinocytes plus the 2 ml of media and then let fall drop-wise back onto the gel. Shaking or rocking the plate may result in uneven distribution of cells to one side of the collagen gel. Place the plate in the 37&deg;C incubator.</li>
	<li>The cells should be grown to confluency, replacing the E medium daily. The cells are confluent when the medium changes to yellow one day after a change in medium. This usually takes 2-4 days. If the medium has not changed by day four, proceed to the next step.</li>
</ol>
4. Making the Raft Cultures
<ol>
	<li>Use sterile forceps to place a sterile metal raft grid, bent sides down, into a 10 cm dish. Aspirate the medium from collagen gel. To loosen the gel from the sides of the well, go around the perimeter of the gel with a sterile spatula using an up and down motion. To remove the collagen gel, tilt the plate slightly and lift by placing a spatula underneath. Lay the collagen gel on the metal grid without generating bubbles between the grid and the gel. Two gels can be placed on each grid.</li>
	<li>In order to make an air-liquid interface, add E medium without EGF slowly to the bottom of the dish so the raft grid is touching the media but the collagen gel is not. The media should not come through the holes in the grid. Add the medium slowly to avoid generating bubbles under the grid, as this will prevent uniform differentiation. Incubate the rafts at 37&deg;C and change the medium every other day maintaining the air-liquid interface. Harvest the rafts after 14 days.</li>
</ol>
5. Representative Results
When the protocol is performed correctly with primary keratinocytes, this procedure will result in a well-differentiated epithelium where the different layers of the skin are evident. This be achieved through histological examination by staining sections of raft cultures for hematoxylin and eosin (H&amp;E), as shown in Figure 1B. H&amp;E staining is also useful to examine the morphology of rafts and the effect of viruses or antiviral compounds on the ability of cells to stratify and differentiate. For example, rafts made from HPV-immortalized primary human keratinocytes (HFK-31) are notably thicker compared to rafts made from normal human foreskin keratinocytes (HFK), reflecting an increased rate of proliferation and the ability of HPV proteins to maintain differentiating cells active in the cell cycle (Figure 1B)10. This results in retention of nuclei throughout the epithelium, whereas normal keratinocytes exit the cell cycle upon differentiation, resulting in a breakdown of the nuclear envelope. To more closely examine differentiation, immunohistochemistry can be performed to examine specific differentiation markers, including cytokeratins, as well as involucrin, and filaggrin. Different cytokeratins are expressed at specific stages of differentiation7. As shown in Figure 2, cytokeratin 10 (K10) is not expressed in the basal layer, and is only found in the suprabasal layers that consist of cells progressively differentiating. In HPV positive rafts, the expression of K10 is delayed compared to that of normal keratinocytes, again reflecting the influence of HPV proteins on keratinocyte differentiation. Immunohistochemistry can also be used to examine the expression profile of viral genes. Shown in Figure 3 is a representative staining examining the expression of the HPV protein E1^E4, which is a late protein whose expression is restricted to the uppermost layers of the epithelium. As expected, no staining is observed in raft cultures from normal human foreskin keratinocytes.
<img alt="Figure 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> 
Figure 1. A) Outline of method for preparing organotypic raft cultures from primary keratinocytes or epithelial cell lines. B) Hematoxylin and eosin staining of organotypic raft cultures generated from human foreskin keratinocytes stably maintaining HPV-31 (HFK-31) episomes, or from normal human foreskin keratinocytes (HFK). The individual epithelial layers are identified, as well as the collagen plug.
<img alt="Figure 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> 
Figure 2. Cytokeratin 10 (K10) expression is restricted to the suprabasal layers of the epithelium. Immunohistochemistry was performed on cross sections of organotypic raft cultures generated from HFK-31 cells, as well as normal HFKs using an antibody to K10. Cellular DNA was counterstained with DAPI. Images were captured using confocal fluorescence microscopy. Arrows indicate the basal layer of the epithelium.
<img alt="Figure 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> 
Figure 3. The HPV E1^E4 protein is produced late in the productive phase of the viral life cycle. Immunohistochemistry was performed on cross sections of organotypic raft cultures generated from HFK-31 cells, as well as normal HFKs using antibodies to E1&circ;E4. Cellular DNA was counterstained with DAPI. Images were captured using confocal fluorescence microscopy. Arrows indicate the basal layer of the epithelium.

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

We have nothing to disclose.

We describe here a method that can be used to study epithelial differentiation in general, but can also easily be adapted to study viral pathogenesis, as well as the efficacy of potential therapeutics. Many viruses target epithelial cells either as the primary site of infection, as in the case of HPV, or at some point in the viral life cycle, as with herpesviruses. Although growing organotypic raft cultures is time consuming, the ability to faithfully recapitulate in vivo epithelial differentiation provides an e...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Rat Tail Collagen type 1 (4-5mg/ml)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
		</tr>
		<tr>
			<td>DMEM without NaHC03</td>
			<td>Invitrogen</td>
			<td>12100-061</td>
		</tr>
		<tr>
			<td>NaHC03</td>
			<td>Sigma</td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Calbiochem</td>
			<td>391338</td>
		</tr>
		<tr>
			<td>Stainless Steel metal grids</td>
			<td>Williams and Mettle Co.</td>
			<td>CR-03063-040-100-S</td>
		</tr>
		<tr>
			<td>E medium11</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>BD Biosciences</td>
			<td>354010</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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 ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.1K visualizações.  University of North Carolina-Chapel Hill. Um In vitro Método para simular In vivo Diferenciação epitelial é descrito. Muitos vírus alvo células epiteliais, como parte do seu ciclo de vida do vírus, e este método proporciona um meio de análise de vírus: interacções hospedeiras que mais se assemelha a que ocorre In vivo. Esta técnica pode ser usada com queratinócitos primários, linhas celulares estabelecidas, bem como normais ou doentes de tecidos de bióp...

Vídeo: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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.

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...