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EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b Immunopanning (Day 0)
NOTE: Prepare 1 immunopanning dish for every 1 - 2 juvenile rat brains.
<ol>
	<li>Add 25 mL of 50 mM Tris pH 9.5 solution to a 15-cm Petri dish.</li>
	<li>Add goat anti-mouse IgG (H+L chains) to the dish for a final concentration of 6 &#181;g/mL. Swirl plate to evenly distribute.</li>
	<li>Incubate the dish for 1 - 3 h at 37 &#176;C.</li>
	<li>Rinse dishes three times with DPBS++ (phosphate-buffered saline (PBS) with Ca2+ and Mg2+), then replace with a solution of panning buffer containing 1 &#181;g/mL OX42 antibody. Leave the dishes overnight at room temperature on a flat surface.</li>
</ol>
2. Tissue Collection (Day 1)
NOTE: This protocol should take ~3 - 4 h.
<ol>
	<li>Before beginning, ensure all solutions are sterile and chilled on ice. Chill all instruments on ice and sterilize with ethanol prior to use.</li>
	<li>Following appropriate regulatory procedures, sacrifice a juvenile laboratory rat by carbon dioxide asphyxiation. 
	NOTE: Alternatively, younger animals may be sacrificed with a lethal dose of ketamine/xylazine (100 - 200 &#181;L of 24 mg/mL ketamine, 2.4 mg/mL xylazine). Ketamine/xylazine must be used if animals are less than 14 days of age. If using multiple animals, extract the tissue from one animal and place it into pre-chilled DPBS++ on ice before proceeding to subsequent animals.</li>
	<li>Pinch a hindpaw and ensure complete unresponsiveness from the animal before proceeding.</li>
	<li>Transcardially perfuse the animal with 10 - 30 mL of ice-cold perfusion buffer using a 27&#189;-G needle until buffer runs clear. 
	NOTE: Volume of Perfusion buffer will vary depending on the size/age of the animal.</li>
	<li>Immediately after perfusion remove the head of the animal. Coming in from the spinal cord cut the occipital condyle on each side with dissection scissors, be careful not to damage the brain. After cutting each side carefully, cut up one side along the parietal and frontal bone towards the nasal bone. With forceps carefully pull back the top of the skull, quickly remove the brain (or CNS structures of interest), and place into pre-chilled DPBS++ on ice.</li>
	<li>Repeat for all remaining animals. 
	NOTE: All proceeding steps should be performed in a laminar flow hood under proper sterile conditions.</li>
</ol>
3. Mechanical Dissociation (Day 1)
<ol>
	<li>After all brains have been collected, chop one brain into 1 mm3 chunks in a Petri dish on ice with a cold scalpel blade, and transfer to an ice-cold dounce homogenizer with 5 - 7 mL ice-cold douncing buffer. Dissociate one brain at a time.</li>
	<li>Dissociate the tissue using 10 - 20 gentle and incomplete strokes with a loose-fitting dounce homogenizer. Take care not to directly crush the tissue at the bottom of the homogenizer, but instead impel the tissue through the space between the sides of the piston and the homogenizer.</li>
	<li>Carefully remove the piston to prevent introduction of air bubbles. Allow poorly dissociated tissue chunks to settle to the bottom of the homogenizer, and transfer supernatant to a new chilled 50-mL conical tube.</li>
	<li>Replace the removed volume with fresh douncing buffer, and repeat steps 3.2 and 3.3 for a total of 3 - 4 rounds, or until all tissue has been dissociated. Repeat the procedure for each brain.</li>
</ol>
4. Myelin Removal (Day 1)
NOTE: Myelin removal is used for isolation of microglia from animals older than P12.
<ol>
	<li>Measure the volume of the cell suspension in the 50-mL conical tube using a 25 mL pipette, then add ice-cold douncing buffer to adjust the total volume to 33.5 mL.</li>
	<li>Add 10 mL of myelin separation buffer (MSB) to the cell suspension and mix thoroughly by inverting the tube several times. This will result in a 23% final concentration of MSB in a volume of 43.5 mL.</li>
	<li>Centrifuge cells for 15 min at 500 x g at 4 &#176;C with slow braking. 
	NOTE: The centrifuge should take approximately 1.5 - 2 min to decelerate. This will generate an upper layer of myelin and dead cell debris, a somewhat murky supernatant, and a smaller pellet that is enriched for live cells.</li>
	<li>Remove the top layer of myelin/debris and the supernatant with a pipette. Take care when removing the top layer to ensure as much of it is removed as possible.</li>
	<li>Resuspend the cell pellet in 12 mL of panning buffer. Gently triturate the cell suspension to break up any clumps of cells that might remain.</li>
</ol>
5. Immunopanning (Day 1)
<ol>
	<li>Pass the cell suspension through a 70-&#181;m cell strainer to remove large debris or cell clumps.</li>
	<li>Rinse the OX42-coated panning dish three times with DPBS++. Don&#39;t allow the plate to completely dry between washes.</li>
	<li>Pour off the last DPBS++ wash and apply the filtered cell suspension to the panning dish. Gently swirl the plate to distribute the cells, then incubate the plate on a flat surface at room temperature for 20 min to allow cells to adhere. Do not incubate longer than 20 min or cells will become very difficult to recover from the dish.</li>
	<li>Rinse the panning dish with DPBS++ 10 times to remove non-adherent cells. Microglia will be firmly attached to the plate, so swirl the plate with each rinse to ensure removal of other non-adherent cells.</li>
	<li>Pour off the last DPBS++ wash and replace with 15 mL DPBS++ and 200 &#956;L trypsin (1.25% stock solution).</li>
	<li>Incubate the dish for &#8804;10 min at 37 &#176;C with 10% CO2 to trypsinize. 
	NOTE: Do not continue longer than 10 min or microglia will become difficult to remove.</li>
	<li>After 10 min of trypsinization, microglia will still be stuck to plate. Pour off trypsin/DPBS++ and gently wash 2x with DPBS++ to remove trypsin, replace with 12 mL of ice-cold microglia growth medium (MGM).</li>
	<li>Place panning dish on ice for 2 min to help weaken cell/substrate interaction, and make sure the dish is flat to prevent areas of the panning dish from drying out.</li>
	<li>Pipette vigorously with a 10-mL pipette and pipet controller on high speed to recover cells from the panning dish. Draw a 16 x 16 grid with the stream of liquid from the pipette to try and remove all the cells.</li>
	<li>Check cells under a microscope at 20X magnification to make sure cells have detached from plate. Mark spots on top of the dish where cells are still stuck, and repeat pipetting in those areas.</li>
	<li>Collect cell suspension and aliquot 3 - 4 mL of supernatant per 15-mL conical tube. Spin for 15 min at 500 x g at 4 &#176;C with slow braking. 
	NOTE: Spinning microglia through a small volume allows for the maximum recovery of cells.</li>
	<li>Aspirate the supernatant, leaving 0.5 mL of MGM with the cell pellet.</li>
	<li>Resuspend each pellet in remaining MGM, and pool the cells from all the tubes.</li>
	<li>Count cells with hemocytometer.</li>
	<li>Plate cells as described in Steps 6 - 7 depending on the application.</li>
	<li>Culture cells at 37 &#176;C with 10% CO2. 
	NOTE: Cells can be culture for up to 3 - 4 weeks with regular media changes or one week with no media changes.</li>
</ol>
6. Spot Coating Tissue Culture Plates/Coverslips (Day 1)
<ol>
	<li>Plate 15 &#181;L of collagen IV coating directly in the center of a 24-well anionic/cationic coated tissue culture plate (see Table of Materials for details) and incubate for 15 min at 37 &#176;C with 10% CO2.</li>
	<li>After counting cells with hemocytometer dilute cells to 2.3 x 105/mL in MGM. Aspirate collagen IV spot and immediately plate 15 &#181;L of cell suspension to this spot; this will give 3.5 x 103 cells/spot. Incubate for 5 - 10 min at 37 &#176;C with 10% CO2 to allow cells to adhere, add 500 &#181;L of CO2-equilibrated TGF-&#946;2/IL-34/cholesterol containing growth medium (TIC) gently to the well.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Douncing Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 200 &#956;L of 0.4% DNaseI in 50 mL of DPBS++ 
			Comments: Use when ice-cold</td>
		</tr>
		<tr>
			<td>Panning Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 2 mg/mL of peptone from milk solids in DPBS++</td>
		</tr>
		<tr>
			<td>Microglia Growth Medium (MGM)</td>
			<td></td>
			<td></td>
			<td>Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 2 mM glutamine, 5 &#956;g/mL N-acetyl cysteine, 5 &#956;g/mL insulin, 100 &#956;g/mL apo-transferrin, and 100 ng/mL sodium selenite 
			Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.</td>
		</tr>
		<tr>
			<td>Collagen IV Coating</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing 2 &#956;g/mL collagen IV</td>
		</tr>
		<tr>
			<td>Myelin Seperation Buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 &#956;L 1 M CaCl2, 5 &#956;L 1 M MgCl2 
			Comments: Mix well after the addition of CaCl2 and MgCl2</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>&#160;T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>21041-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td></td>
			<td></td>
			<td>Reconstitution: 200 &#956;g/mL in PBS 
			Concentration used: 1:100 
			Storage: -80&#176;C</td>
		</tr>
		<tr>
			<td>mouse anti-rat CD11b monoclonal (clone OX42)</td>
			<td>Bio-Rad</td>
			<td>MCA275R</td>
			<td>Panning: 1:1,000; Staining: 1:500</td>
		</tr>
		<tr>
			<td>TGF-b2/IL-34/Cholesterol containing growth media (TIC)</td>
			<td></td>
			<td></td>
			<td>Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 &#956;g/mL ovine wool cholesterol, 10 &#956;g/mL heparan sulfate, 0.1 &#956;g/ml oleic acid, and 0.001 &#956;g/ml gondoic acid 
			Comments: Make sure to add cholesterol to media warmed to 37 &#176;C and do not add more than 1.5 &#956;g/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.</td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>GE Healthcare</td>
			<td>Cat# 17-5445-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Primaria Plates</td>
			<td>VWR</td>
			<td>Cat# 62406-456</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Worthington</td>
			<td>Cat# DPRFS</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of microglia from rat brains using serum-free media

Source: Collins, H. Y., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57122">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> J. Vis. Exp. (2018)
This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b ...

Take a perfused rat brain. Mince it into small fragments.
Homogenize the tissue in a buffer to release cells, including microglia.
Once the undigested tissue settles, collect the supernatant containing cells and add a myelin separation buffer.
Mix the contents and centrifuge to separate cells from myelin and debris. Remove the myelin-debris layer and supernatant.
Resuspend cells in buffer and pipette to separate clumps.
Pass the suspension through a strainer to remove remaining cell aggregates.
Transfer the filtered cells to a culture plate coated with microglia-specific monoclonal antibodies. Incubate to facilitate microglia adhesion.
Wash with buffer to remove non-adherent cells. Add trypsin and incubate to loosen microglia.
Remove trypsin.
Add serum-free media. Incubate on ice to weaken cell-substrate interactions.
Pipette and collect the suspension in a tube. Centrifuge and remove the supernatant.
Resuspend microglia in serum-free media and transfer onto a collagen-coated multi-well plate. Incubate to facilitate microglia adhesion.
Add media with growth factors and lipids. Incubate for microglia maturation.

isolation-culture-microglia-from-rat-brains-using-serum-free

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

97 次观看. 来源:Collins， H. Y.， et al.， 啮齿动物小胶质细胞的分离和培养在无血清培养基中促进动态分叉形态。J. Vis. Exp. （2018） 该视频演示了一种从大鼠大脑中分离和培养小胶质细胞的方法。它需要使用包被有小胶质细胞特异性单克隆抗体的板将小胶质细胞与其他神经元和非神经元细胞分离。 

视频: 使用无血清培养基从大鼠脑中分离和培养小胶质细胞 - 概念

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.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

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

科研

JoVE Journal

癌症研究

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

神经科学

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

成績單

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media