eoe sub articles

EoE Sub Articles

1. Culture Preparation of Dissociated Adult dorsal root ganglion Neuron or DRG Neuron
<ol>
	<li>Before setting up the culture, pre-coat a 6-well plate with poly-D-lysine and laminin. Incubate a 6-well plate with 0.01% poly-D-lysine at 37&#176;C for 2 h or at 4&#176;C overnight. Then, wash the plate twice with distilled water.</li>
	<li>Incubate the plate with laminin solution at a concentration of 3 &#181;g/mL for 2 h at room temperature, and then wash the plate twice with distilled water. Dry the plate at room temperature for at least 1 h.</li>
	<li>Euthanize a mouse in a transparent CO2 chamber connected to a compressed CO2 gas cylinder. Incise the skin overlying the vertebral column with a surgical blade and dissect the paravertebral muscles bilaterally to expose the vertebral bones. Remove the vertebral bones meticulously using a narrow-tipped surgical rongeur until the DRGs are fully exposed. 
	NOTE: Adult DRG neurons are obtained from adult C57BL6 male mice aged between 8 weeks and 12 weeks old.</li>
	<li>Remove the DRGs bilaterally from the S1 all the way up to the C1 level using iridectomy scissors and fine-tipped forceps under a dissecting microscope. Try to cut out the roots entirely from DRGs in order to minimize the contamination of Schwann cells or fibroblasts.</li>
	<li>Store the dissected DRGs in a 60 mm Petri dish containing 5 mL of cold Dulbecco&#39;s Modified Eagle Medium (DMEM) on ice until all the DRGs are the next step, even if there are still remaining DRGs are collected. 
	NOTE: It is critical to dissect the DRGs as quickly as possible. The collection of all DRGs from one mouse should not take longer than 30 min. 30 min after the euthanasia, stop collecting the DRGs and proceed to Transfer the DRGs to a 1.5 mL Eppendorf tube using a blue pipette tip (1000 &#181;L) with a cut-off end. Remove the DMEM after a quick spin for several seconds using a mini centrifuge. Add 1 mL of DMEM containing 125 U/mL type XI collagenase and incubate the tube for 90 min with a gentle rotation (35-40 rpm) using a twister shaker in a 37 &#176;C incubator. Immobilize the tube on the shaker floor using the tape.</li>
	<li>Discard collagenase-containing DMEM and add 1 mL of fresh DMEM. Wait until the floating DRGs completely sink down to the bottom, and then remove the DMEM with the tissue debris. Repeat this washing step at least six times. 
	NOTE: Do not try to discard the supernatant completely. Be careful not to remove any DRGs with the supernatant DMEM.</li>
	<li>Transfer the DRGs to a 15 mL conical tube using a blue pipette tip (1000 &#181;L) with a cut-off end. Then pipette up and down gently at least 15 times using a blue tip (1000 &#181;L) to make a homogeneous cell suspension. Avoid making bubbles and try not to touch the bottom of the conical tube with a pipette tip.</li>
	<li>Centrifuge the tube at 239 x g for 3 min and carefully discard the supernatant with floating debris. Add 1 mL of Neurobasal medium supplemented with B27 (2.0% v/v) and resuspend the cell pellet by gently pipetting up and down 5 to 10 times.</li>
	<li>Pass the cell suspension through a 70-&#181;m cell strainer overlaid on top of a 50 mL conical tube. Wait for 2 min, and then pour Neurobasal/B-27 medium onto the strainer using a pipette controller.</li>
	<li>Plate all collected DRG neurons ( approximately 2 x 106 DRG neurons from one mouse) onto two wells of 6-well plate. DRG neurons from one adult mouse cover two wells in a 6-well plate. Then place the plate in a CO2 incubator at 37&#176;C until the macrophage co-cultures.</li>
</ol>
2. Co-culture of Primary Peritoneal Macrophages on A Cell Culture Insert
NOTE: Establish the co-cultures 4 h after the initial plating of the dissociated DRG neurons
<ol>
	<li>Primary peritoneal macrophages are prepared from adult C57BL6 male mice aged between 8 weeks to 12 weeks old. Euthanize an animal in a CO2 chamber. Incise the abdominal skin delicately, expose the peritoneum, and avoid cutting the peritoneum to prevent leakage of lavage fluid.</li>
	<li>Puncture the peritoneum using a syringe with a 22G needle and inject 10 mL of ice-cold phosphate-buffered saline (PBS) into the peritoneal cavity. Gently massage the peritoneum for 1-2 min. Then, pull out the needle and squeeze out the PBS through the needle puncture site, and collect the lavage fluid in a 50 mL conical tube. 
	NOTE: Before use, put the syringe on ice to maintain the coldness of the PBS.</li>
	<li>Centrifuge the lavage fluid at 239 x g for 10 min at 4 &#176;C to pellet the cellular components. Resuspend the pellet with 3 mL of the red blood cell (RBC) lysis buffer for 3 min at room temperature. Then centrifuge the cell suspension again at 239 x g for 10 min at 4 &#176;C. Resuspend the pellet with 3 mL of PBS and centrifuge the cell suspension again to remove any remaining RBC lysis buffer. 
	NOTE: Make sure the RBC lysis buffer is completely removed. Otherwise remaining RBC lysis buffer may be toxic to the cultured macrophages.</li>
	<li>Resuspend the pelleted cells in 1 mL of Neurobasal/B-27 medium. Plate half of all collected macrophages (a total of 3 &#215; 106 to 6 x 106 cells) on a cell culture insert with an effective area of 4.2 cm2, which is placed on top of the well of dissociated DRG. 
	NOTE: During the co-culture period, macrophages are grown in the Neurobasal/B-27 neuron culture medium. Adequate survival of macrophages under this condition was confirmed.</li>
</ol>
3. Treatment of db-cAMP (dibutyryl-cyclic AMP)&#160; and Collection of Macrophage CM
NOTE: Start db-cAMP treatment 4 h after the neuron-macrophage co-cultures.
<ol>
	<li>Add 2 &#181;L of 100 &#181;M db-cAMP solution to the neuron-macrophage co-cultures. Add the same volume of PBS for a control experiment.</li>
	<li>After 24 h, fill an empty well with 1 mL of macrophage culture medium in the same 6-well plate. Transfer the cell culture insert in the neuron-macrophage co-cultures to the empty well with a macrophage culture medium. Keep the cells under the same condition for 72 hours without changing the medium. 
	NOTE: We add only 1 mL of macrophage culture medium during CM collection to make concentrated CM. During the CM collection, if needed, we added more to completely cover the macrophages within the insert.</li>
	<li>After 72 h, centrifuge the macrophage CM at 239 x g for 5 min to remove the cellular components. Pass the supernatant through a 0.2-&#181;m filter to remove any remaining cellular debris. Store the collected CM at -70 &#176;C until use.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture insert transparent PET membrane 0.4&#956;m pore size&#160;&#160;&#160;</td>
			<td>Corning,Falcon</td>
			<td>353090</td>
			<td>Transparent PET membrane with 0.4-&#956;m pore size, for 6-well plate</td>
		</tr>
		<tr>
			<td>70-&#956;m nylon cell strainer&#160;</td>
			<td>Corning, Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer&#160;</td>
			<td>Qiagen</td>
			<td>158904</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium&#160;&#160;&#160;</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>21103-049</td>
			<td>Containing 1% glutamax and 1% penicilin-streptomycin</td>
		</tr>
		<tr>
			<td>Poly-D-lysine Hydrobromide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Thermo Fisher Scientific,&#160;&#160;</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 3&#39;, 5&#39;-cyclic monophosphate, N6 ,O2&#39;-dibutyryl-, sodium salt&#160;</td>
			<td>Merck Millipore Corporation, Calbiochem</td>
			<td>28745</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture CO2 &#160;incubator&#160;</td>
			<td>Panasonic</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

developing a neuron and macrophage coculture in vitro

Source:Yun, H. J., et al. <a href="https://app.jove.com/v/56920">Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity.</a> J. Vis. Exp. (2018).
This video shows the co-culturing of murine dorsal root ganglion neurons and macrophages. Neurons are seeded on a coated plate, while macrophages are collected from an euthanized mouse, treated, and added to the culture insert. Treatment with db-cAMP activates neurons and prompts macrophages to adopt a pro-regenerative phenotype to support neuronal outgrowth.

Developing a Neuron and Macrophage Coculture In Vitro

1. Culture Preparation of Dissociated Adult dorsal root ganglion Neuron or DRG Neuron

	Before setting up the culture, pre-coat a 6-well plate with ...

Begin with isolated murine dorsal root ganglion neurons in a growth medium.
Seed them onto a polymer-coated multi-well plate to promote neuronal attachment.
Next, take a dissected euthanized mouse and inject ice-cold PBS into its peritoneal cavity.
Gently massage the peritoneum to dislodge macrophages from the peritoneum wall.
Squeeze the mouse to collect the peritoneal fluid and treat it with a lysis buffer to selectively lyse the red blood cells, which appear as contaminants. Centrifuge and resuspend the cells in a culture medium.
Transfer the macrophages to a porous insert positioned within the well containing the cultured neurons.
This co-culture allows neurons and macrophages to be in close proximity.
Treat the co-culture with db-cAMP and incubate to facilitate its entry into the cells. This activates the neurons to secrete the signaling molecules.
These signaling molecules, along with db-cAMP, induce macrophages to adopt a pro-regenerative phenotype and secrete essential factors for neuronal outgrowth.

developing-a-neuron-and-macrophage-coculture-in-vitro

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

131 Views. Źródło:Yun, H. J., et al. Neuro-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity. J. Vis. Exp. (2018). Ten film pokazuje współhodowlę neuronów zwojowych i makrofagów mysiego korzenia grzbietowego. Neurony wysiewa się na powlekanej płytce, podczas gdy makrofagi są pobierane od myszy poddanej eutanazji, poddawane obróbce i dodawane do wkładki hodowlanej. Leczenie db-cAMP aktywuje neurony i skłania makrofagi do przyjęcia feno...

Video: Opracowanie kokultury neuronów i makrofagów in vitro - Concept

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

JoVE Journal

Cancer Research

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Developing a Neuron and Macrophage Coculture In Vitro

Transkrypcja

Developing a Neuron and Macrophage Coculture In Vitro