The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Tel-Aviv University.
1. Rat Protocol
<ol>
	<li>Preparations
	<ol>
		<li>Arrange 2 butterfly 21 G needles. Optionally, prepare 2 blunt-edged butterfly 21 G needles by filing the sharp edge of the needle.</li>
		<li>Sterilize surgical tools: 2 pairs of scissors, blunted-edged forceps, hemostat, tooth-tissue forceps sterilized by autoclave at 121 &#176;C for at least 30 min on gravity (dry) setting.</li>
		<li>Prepare heparinized PBS (30 units/ml) by adding 30 units of heparin per ml of phosphate buffered saline (1x PBS) solution. Use at RT. 35 ml is the minimal volume needed per animal.</li>
		<li>Stream heparinized PBS into the peristaltic pump lines and the butterfly needles to avoid air bubbles.</li>
	</ol>
	</li>
	<li>Perfusion of the Lungs and Collection of MP-leukocytes
	<ol>
		<li>Euthanize the animal by an overdose of 8% isoflurane. Confirm euthanasia by monitoring breathing cessation.</li>
		<li>Upon cessation of respiration, immediately open the peritoneal and chest cavities using the sterile scissors and tooth-tissue forceps. Perform a midline abdominal incision along the abdomen up to the xiphoid process.</li>
		<li>Lift the sternum using the forceps, cautiously cutting the rib cage on both sides, without puncturing any internal organ or large blood vessels. Clamp a hemostat on the sternum, and rostrally fold the rib cage to expose the cardiopulmonary complex.</li>
		<li>Insert a butterfly needle into the right ventricle of the heart, and within approximately a minute collect the maximal volume of blood into a syringe (~6 ml from a 250 g animal).</li>
		<li>Clamp the vena cava with a hemostat to avoid backward perfusion of the liver (see Discussion).</li>
		<li>Hold the butterfly needle within the right ventricle while replacing the syringe containing the blood with the outflow pipe of the peristaltic pump.</li>
		<li>Insert a second butterfly needle connected to a 5 ml harvesting syringe into the left ventricle, avoiding penetration of the interventricular septum.</li>
		<li>Turn on the peristaltic pump at a speed of approximately 5 ml/min and gently collect into the syringe the first milliliters of perfusate that are contaminated with blood. Continue until the perfusate color turns from dark to pale red.&#160;(Discard the blood-contaminated perfuste).</li>
		<li>Without cessation of the peristaltic pump, rapidly replace the 5 ml harvesting syringe with a 20 ml harvesting syringe and collect 20 ml of lung perfusate employing a higher speed of perfusion (up to 7 ml/min). Continuously monitor the perfusate flow into the collecting syringe, while avoiding vacuum formation.</li>
		<li>Cease the flow of the peristaltic pump.</li>
	</ol>
	</li>
	<li>Leukocytes Extraction
	<ol>
		<li>Centrifuge the perfusate for 10 min at 400 &#215; g.</li>
		<li>Aspirate the fluid.</li>
		<li>Add 10 ml PBS or medium, centrifuge at 400 x g for 10 min, and aspirate the fluid. Repeat this step twice. The use of PBS or medium depends on the consequent use of the sample.</li>
		<li>Add 20 ml PBS or medium, centrifuge at 400 x g for 10 min, and aspirate the fluid.</li>
		<li>Reconstitute the cells to the desired concentration, based on the sample usage (FACS, PCR, etc.).</li>
	</ol>
	</li>
</ol>
2. Mouse Protocol
<ol>
	<li>Preparations
	<ol>
		<li>Arrange 2 butterfly 25 G needles. Optionally, prepare 2 blunt-edged butterfly 25 G needles by filing the sharp edge of the needle.</li>
		<li>Sterilize surgical tools: 2 pairs of scissors, blunted-edged forceps, hemostat, tooth-tissue forceps sterilized by autoclave at 121 &#176;C for at least 30 min on gravity (dry) setting.</li>
		<li>Prepare heparinized PBS (30 units/ml) by adding 30 units of heparin per ml of 1x PBS solution. Use at RT. 25 ml is the minimal volume needed per animal.</li>
		<li>Stream heparinized PBS into the peristaltic pump lines and the butterfly needles to avoid air bubbles.</li>
	</ol>
	</li>
	<li>Perfusion of the Lungs and Collection of MP-leukocytes
	<ol>
		<li>Euthanize the animal by an overdose of 8% isoflurane. Confirm euthanasia by monitoring breathing cessation.</li>
		<li>Upon cessation of respiration, immediately open the peritoneal and chest cavities using the sterile scissors and tooth-tissue forceps. Perform a midline abdominal incision along the abdomen up to the xiphoid process.</li>
		<li>Lift the sternum using the forceps, cautiously cutting the rib cage on both sides, without puncturing any internal organ or large blood vessels. Clamp a hemostat on the sternum, and rostrally fold the rib cage to expose the cardiopulmonary complex.</li>
		<li>Clamp the vena cava with a hemostat to avoid backward perfusion of the liver(see Discussion).</li>
		<li>Insert a butterfly needle connected to the outflow pipe of the peristaltic pump into the right ventricle of the heart, and a second butterfly needle connected to a 2 ml harvesting syringe into the left ventricle of the heart, avoidingpenetration of the interventricular septum.</li>
		<li>Turn on the pump to a speed of approximately 2 ml/min and collect the first milliliters of blood-contaminated perfusate into the harvesting syringe until the perfusate turns from dark to pale red (Discard the blood-contaminated perfuste).</li>
		<li>Without cessation of the peristaltic pump, rapidly replace the 2 ml harvesting syringe with a 10 ml harvesting syringe and collect 10 ml of lung perfusate employing a higher speed of perfusion (up to 4 ml/min). Continuously monitor the perfusate flow into the collecting syringe, while avoiding vacuum formation.</li>
		<li>Cease the flow of the peristaltic pump.</li>
	</ol>
	</li>
	<li>Leukocytes Extraction
	<ol>
		<li>Centrifuge the perfusate for 10 min at 400 &#215; g.</li>
		<li>Aspirate the fluid.</li>
		<li>Add 10 ml PBS or medium, centrifuge at 400 x g for 10 min, and aspirate the fluid. Repeat this step three times. The use of PBS or medium depends on the consequent use of the sample.</li>
		<li>Add 10 ml PBS or medium, centrifuge at 400 x g for 10 min, and aspirate the fluid.</li>
		<li>Reconstitute the cells to the desired concentration, based on the sample usage (FACS, PCR, etc.).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, J. 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+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

The authors have nothing to disclose.

The method presented herein enables the selective harvesting and studying of the unique population of MP-leukocytes. Compared to circulating or splenic leukocytes, the MP population is characterized by a distinct composition of leukocyte subpopulations, higher activation levels, higher release of various cytokines, and higher mRNA levels of pro- and anti-inflammatory cytokines2,3. Specifically, we have shown that MP-NK cells are more cytotoxic than circulatory NK cells against various target cells3,4</sup...

Leukocytes adhering to the capillaries of the lungs (i.e., marginating-pulmonary (MP) leukocytes)1 were shown to exhibit distinct leukocyte composition and unique activity compared to leukocytes from other immune compartments (e.g., circulation, spleen, bone-marrow)2-4. For example, MP-leukocytes exhibit higher natural killer (NK) cells cytotoxicity against various tumor cells, compared to circulating and splenic NK cells, as well as differentiated messenger RNA (mRNA) levels and increased secretion of pro- and anti-inflammatory cytokines. The composition of cells is also differentiated from circulating leukocytes as MP-leukocytes have a higher ratio of innate/adaptive immunity compared to circulating leukocytes (50% vs. 30%, respectively). The goal of the method presented herein is to enable selective harvesting of MP-leukocytes, in order to study this important and unique immune compartment (cell population), and to elucidate the impact of various manipulations (e.g., immune activation) on these specific cells.
To understand the significance of this unique population, it is important to note that the immune system can control circulating tumor cells, micrometastases, and residual disease through in vivo functions of cell-mediated immunity (CMI). This ability is evident despite the precedent failure of the immune system to control the primary tumor, and supported by ample in vivo evidence in cancer patients and animal models5. Importantly, these findings are often inconsistent with in vitro and ex vivo studies, which report that most autologous tumor cells are resistant to cytotoxicity by circulating leukocytes in blood samples from humans and animals (measured by cytotoxicity assays) 6,7. This discrepancy may be attributed to the in vivo existence of distinct leukocyte populations, such as the aforementioned MP leukocyte population, and specifically its subpopulation of activated NK cells3. Indeed, syngeneic tumor cells (MADB106), which were found to be resistant to circulating and splenic leukocytes, were shown to be lysed by MP-NK cells3,8. Thus, the allegedly &#39;NK-resistant&#39; MADB106 cells that metastasize to lungs of fischer344 (F344) rats are controlled by MP-NK cells, but not by circulating or splenic NK cells, which are commonly studied given their ease of access.
Purified and active MP-leukocytes are inaccessible through the standard harvesting methods of leukocytes from the lungs, which are based on lung tissue grinding or biological degradation9. Our approach has two major advantages compared to tissue processing approaches. First, the perfusion approach selectively harvests MP-leukocytes, separating them from other cells that originate from the lung parenchymal, interstitial, and broncho-alveolar compartments. Second, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, unlike the grinding and biological processing approaches that damage cells, alter their morphology, and induce the production and release of various factors that modulate immune activity and specifically suppress NK cytotoxicity10.
The lungs are a major target organ for cancer metastasis and for various infectious diseases. All circulating malignant cells and infected cells pass through the lung capillaries, where they need to deform and interact with capillary endothelial cells and resident leukocytes. Under these conditions, circulating cells can be easily targeted by resident MP-leukocytes. It thus seems biologically advantageous to have activated leukocytes in this immune compartment, and it is important to study this unique MP-population in different biological, experimental, and clinical settings. It is worthy to note that systemic immune activation by various biological-response-modifiers (e.g., polyinosinic-polycytidylic acid (poly I:C) or type-C CpG oligodeoxynucleotides (CpG-C ODN)) have been shown to activate MP-leukocytes more than circulating leukocytes3,8,11.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

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.

The MP-compartment exhibit a different leukocyte subset composition compared to circulating leukocytes. Using flow cytometry analysis, leukocyte subpopulations were identified and quantified to characterize the composition of both circulating and MP leukocytes. Granulocytes and lymphocytes were identified based on forward and side scatters. Within the lymphocytes, NKRP-1bright cells were identified as NK cells, CD3+ as T cells, RM1+ as monocytes, and CD4<s...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

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 ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K Views.  Tel-Aviv University. 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.

Video: Selective Harvesting of Marginating-pulmonary Leukocytes

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

Medicine

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Establishing In Vitro LLEC Cultures Using Magnetic Cell Selection

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Author Spotlight: Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Neuroscience

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although ...

Biology

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://www.jove.com/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...

Selective Harvesting of Marginating-pulmonary Leukocytes

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