eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preparation of Reagents
<ol>
	<li>Digestion buffer (1 ml per hemisphere): Dissolve a standardized mixture of purified digestive enzymes, e.g., Liberase with low thermolysin concentration (TL) to a concentration of 2U/ml in Hanks Balanced Salt Solution (HBSS) containing calcium (Ca) and magnesium (Mg).</li>
	<li>Washing buffer with DNAse: Dissolve DNAse I to a concentration of 666U/ml in HBSS (Ca/Mg (calcium/magnesium) free) containing 10% of fetal calf serum (FCS).</li>
	<li>Washing buffer without DNAse: HBSS (Ca/Mg free) containing 10% of FCS.</li>
	<li>Density gradient medium (5 ml per hemisphere): prepare stock isotonic density gradient medium (SIP; 100%) by mixing nine parts of density gradient medium with one part of 1.5 M sodium chloride. Dilute SIP to 25% density by adding an appropriate volume of HBSS (Ca/Mg free) containing 3% of FCS.</li>
</ol>
2. Dissociation of Cerebral Tissue into Single Cell Suspensions
<ol>
	<li>Carefully remove brain stem and cerebellum with a clean razor blade. Use a clean razor blade to hemisect the brain and cut each hemisphere along the coronal plane into three pieces of roughly equal size.</li>
	<li>Mince dissected tissue of each hemisphere through a 100 &#181;m cell strainer using the plunger end of a 5 ml syringe. Continuously rinse the cell strainer with ice-cold HBSS (+Ca/Mg). Store homogenized samples on ice.</li>
	<li>Centrifuge at 286 x g and 4&#176; C for 5 min, carefully discard the supernatant. Resuspend the pellet in 1 ml of digestion buffer and transfer the suspension into a 2 ml tube. Incubate suspension under slow continuous rotation at 37 &#176;C for 1 hr.</li>
	<li>Sieve the cell suspension through a 70 &#181;m cell strainer and rinse thoroughly with 3 ml of washing buffer containing DNAse followed by 15 ml of DNAse-free washing buffer. Centrifuge at 286 x g and 18 &#176;C for 5 min and discard the supernatant. Note: The use of DNAse eliminates DNA mucus caused by cell lysis, which might lead to cell aggregation, compromising cell survival.</li>
</ol>
3. Density Gradient Centrifugation for Removal of Myelin and Cell Debris
<ol>
	<li>Resuspend the cell pellet in 5 ml of 25% density gradient medium and pipette the suspension to a 15 ml tube. Mix thoroughly by repeated and gentle pipetting, avoiding bubble formation. Note: Density gradient medium should be used at RT to prevent cell clumping.</li>
	<li>Centrifuge at 521 x g and 18 &#176;C for 20 min. Use the lowest acceleration profile of the rotor and allow the rotor to stop without a brake. Gently remove the tubes from the centrifuge without shaking. Carefully aspirate the myelin coat and the supernatant while preserving the cell pellet at the bottom of the tube. Note: Make sure to remove the entire myelin coat, as any leftover will impair flow cytometric analysis.</li>
	<li>Resuspend the pellet in 10 ml of DNAse-free washing buffer and pipette the suspension to a new 15 ml tube. Note: This washing step is crucial since sufficient removal of density gradient medium significantly contributes to the quantity and quality of the final cell sample.</li>
	<li>Centrifuge again at 286 x g and 10 &#176;C for 5 min and discard the supernatant. Resuspend cells in 100 &#181;l of cold washing buffer and determine cell counts and viability by trypan blue exclusion in a hemocytometer. Store samples at 4 &#176;C and further process them quickly.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MACSmix Tube Rotator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Multifuge 3SR+</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s buffered salt solution (HBSS)</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>14175-129</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Calcium/Magnesium</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>24020-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Roche Diagnostics GmbH</td>
			<td>5401020001</td>
			<td>Use at room temperature</td>
		</tr>
		<tr>
			<td>Percoll Plus</td>
			<td>GE Healthcare Europe GmbH</td>
			<td>17-5445-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>PAN Biotech</td>
			<td>3302-P101003</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco/Life Technologies GmbH</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trucount</td>
			<td>BD Biosciences</td>
			<td>340334</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Biochrom AG</td>
			<td>L 182-10</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche Diagnostics GmbH</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Tubing, 1m</td>
			<td>World precision instruments</td>
			<td>503022</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris Scissors sharp/blunt</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight 1x2 teeth forceps</td>
			<td>Fine Science Tools</td>
			<td>11021-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt-end Forceps</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml syringe plunger</td>
			<td>Carl Roth GmbH (Braun)</td>
			<td>EP96.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 100&#181;m</td>
			<td>Dr. I. Schubert, BD</td>
			<td>2360-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican Fine dosage syringe 1ML</td>
			<td>Braun</td>
			<td>TBD</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 70&#181;m</td>
			<td>Greiner Bio-One GmbH</td>
			<td>542 070</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes, 10ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>607180</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes, 10ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>86,12,54,025</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes, 25ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>760180</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes, 5ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>606180</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes,25ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>86,16,85,020</td>
			<td></td>
		</tr>
		<tr>
			<td>serological pipettes,5ml</td>
			<td>Sarstedt AG&#38;Co</td>
			<td>86,12,53,025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips, 0,1-10&#181;l</td>
			<td>Corning B.V.Life Sciences</td>
			<td>4840</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips, 100-1000&#181;l</td>
			<td>Greiner Bio-One GmbH</td>
			<td>740290</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips, 10-200&#181;l</td>
			<td>Greiner Bio-One GmbH</td>
			<td>739296</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes 1,5ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>Reaction tubes 2ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>623201</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological petri dish, 94x16mm</td>
			<td>Greiner Bio-One GmbH</td>
			<td>633180</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15ml</td>
			<td>Greiner Bio-One GmbH</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50ml</td>
			<td>VWR International GmbH (BD)</td>
			<td>734-0448</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer hemocytometer</td>
			<td>Biochrom AG</td>
			<td>PDHC-N01</td>
			<td></td>
		</tr>
		<tr>
			<td>razor blade</td>
			<td>Carl Roth GmbH</td>
			<td>CK07.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining cell suspensions from ischemic mouse brain tissues

Source: P&#246;sel, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53658">Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for isolating brain cells and infiltrated immune cells from ischemic mouse brain tissues by enzymatic digestion, followed by the removal of cell debris and myelin by density gradient centrifugation, to study inflammation responses in stroke research.

Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.
1. Preparation of Reagents

	Digestion ...

Take perfused ischemic mouse brain tissues containing brain cells and infiltrated immune cells.
Mince the tissues on a strainer and rinse with buffer.
Centrifuge the filtrate and discard the supernatant.
Resuspend the pellet in a digestive enzyme solution and transfer it to a microcentrifuge tube.
Incubate for the digestive enzymes to break down the extracellular matrix, loosening the cells.
Filter the suspension and rinse with buffer containing DNase to remove contaminating DNA.
Next, wash with DNase-free buffer.
Centrifuge the filtrate and discard the supernatant.
Resuspend the cells in density gradient medium. Then, transfer the contents to a fresh tube and mix. Centrifuge to separate the components by density.
Remove the supernatant containing myelin and debris.
Resuspend the pellet in DNase-free buffer. Transfer it to a new tube and centrifuge again.
Discard the supernatant to ensure sufficient removal of the density gradient medium.
Finally, resuspend the cells in buffer for further analysis.

obtaining-cell-suspensions-from-ischemic-mouse-brain-tissues

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Cerebrovascular Diseases

38 Views. Source: Pösel, C., et al. Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain. J. Vis. Exp. (2016) This video demonstrates the procedure for isolating brain cells and infiltrated immune cells from ischemic mouse brain tissues by enzymatic digestion, followed by the removal of cell debris and myelin by density gradient centrifugation, to study inflammation responses in stroke research. 

Video: Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues - Concept

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

JoVE Journal

Immunology and Infection

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

Transcript

Obtaining Cell Suspensions from Ischemic Mouse Brain Tissues

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses