eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse Protocol
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	<li>Perfusion of the Liver and Collection of MH-Leukocytes
	<ol>
		<li>Euthanize the animal with an overdose of 8% isoflurane. As a precaution, place a 15 ml tube containing an isoflurane-soaked pad around the mouse&#39;s head until opening its chest cavity.</li>
		<li>Fix the limbs of the mo...

isolation of marginating hepatic leukocytes from a mouse liver

Source: Sorski, L., et al. <a href="https://app.jove.com/v/53918">Selective Harvesting of Marginating-hepatic Leukocytes.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of marginating hepatic leukocytes from mouse liver by forced perfusion.

Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

Source: Sorski, L., et al. Selective Harvesting of Marginating-hepatic Leukocytes. J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of ...

Marginating hepatic, MH, leukocytes, are found adhered to the walls of specialized liver blood vessels or sinusoids, which are lined by endothelial cells. MH leukocytes are recruited to the surrounding tissue in response to inflammation.
To harvest MH leukocytes, begin with a euthanized mouse in a supine position. Incise the peritoneal and chest cavities, exposing the liver and cardiopulmonary complex.
Displace the intestines to expose the portal vein, which carries blood from the spleen, gastrointestinal tract, and its associated organs to the liver. Insert a needle connected to a peristaltic pump into the portal vein.
Dissect the inferior vena cava &#8212; a major blood vessel carrying deoxygenated blood from the liver to the heart &#8212; to collect the perfusate during the perfusion.
Perfuse the liver through the portal vein with buffer containing anticoagulant at a low flow rate for a short duration. This step helps to remove blood with non-adherent cells from the liver&#39;s blood vessels through the inferior vena cava outflow. Discard this blood-contaminated perfusate.
Now, perfuse at a higher flow rate. The forced perfusion at an increased flow and shear rates disrupts the adhesion between the MH leukocytes and blood vessel inner lining, thus detaching the MH leukocytes from the sinusoidal wall.
Collect and transfer the perfusate containing MH leukocytes and other blood cells into a fresh tube. Centrifuge. Resuspend the pellet containing MH leukocytes for further downstream analysis.

isolation-of-marginating-hepatic-leukocytes-from-a-mouse-liver

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

JoVE Journal

Immunology and Infection

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Isolation of Marginating Hepatic Leukocytes from a Mouse Liver

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