Name: characterization of immune cells and proinflammatory mediators in the pulmonary environment
Uploaded: 2020-06-24T12:30:00
Description: Watch this Scientific Journal Video about Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment at JoVE.com

Este trabajo fue apoyado por la subvención P20 GM109098 de los NIH y el Programa de Premios a la Innovación de Praespero a Edwin Wan. Los experimentos de citometría de flujo se realizaron en la WVU Flow Cytometry & Single Cell Core Facility, que cuenta con el apoyo de las subvenciones de los NIH S10 OD016165, U57 GM104942, P30 GM103488 y P20 GM103434.

Todos los protocolos y procedimientos realizados fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de West Virginia. Los ratones fueron alojados en condiciones libres de patógenos específicos en el vivero de la Universidad de Virginia Occidental.
1. Preparación de soluciones
<ol><li>Preparar tampón de perfusión (solución salina tamponada con fosfato, PBS). Use aproximadamente dos alícuotas de 10 ml de PBS helado por ratón.</li>
	<li>...

<ol>
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Y., 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=Ischemic+stroke+alters+immune+cell+niche+and+chemokine+profile+in+mice+independent+of+spontaneous+bacterial+infection.">Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.</a> Immunity, Inflammation and Diseases. 7 (4), 326-341 (2019).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Lehmann, J. S., Zhao, A., Sun, B., Jiang, W., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Cytokine+Profiling+of+Stimulated+Mouse+Splenocytes+Using+a+Cytometric+Bead-based+Immunoassay+Platform.">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> Journal of Visualized Experiments. (129), e56440 (2017).</li><li>Shi, C., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+recruitment+during+infection+and+inflammation.">Monocyte recruitment during infection and inflammation.</a> Nature Reviews Immunology. 11 (11), 762-774 (2011).</li><li>Monaghan, K. L., Zheng, W., Hu, G., Wan, E. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+Monocyte-Derived+Antigen-Presenting+Cells+Have+Distinct+Gene+Signatures+in+Experimental+Model+of+Multiple+Sclerosis.">Monocytes and Monocyte-Derived Antigen-Presenting Cells Have Distinct Gene Signatures in Experimental Model of Multiple Sclerosis.</a> Frontiers in Immunology. 10, 2779 (2019).</li><li>Zhai, X., 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=A+novel+technique+to+prepare+a+single+cell+suspension+of+isolated+quiescent+human+hepatic+stellate+cells.">A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells.</a> Science Reports. 9 (1), 12757 (2019).</li><li>Platzer, 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=Dendritic+cell-bound+IgE+functions+to+restrain+allergic+inflammation+at+mucosal+sites.">Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites.</a> Mucosal Immunology. 8 (3), 516-532 (2015).</li><li>Shinoda, K., 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=Thy1+IL-7++lymphatic+endothelial+cells+in+iBALT+provide+a+survival+niche+for+memory+T-helper+cells+in+allergic+airway+inflammation.">Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation.</a> Proceedings of the National Academy of Sciences U. S. A. 113 (20), 2842-2851 (2016).</li><li>Nakahashi-Oda, C., 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=Apoptotic+epithelial+cells+control+the+abundance+of+Treg+cells+at+barrier+surfaces.">Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.</a> Nature Immunology. 17 (4), 441-450 (2016).</li><li>Barrott, J. 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Los protocolos descritos aquí permiten la identificación de tipos de células inmunes pulmonares y la expresión de quimiocinas o citoquinas en el mismo ratón. Si se desea un estudio histopatológico, se puede extraer y fijar un lóbulo individual para ese propósito antes de proceder a los pasos de aislamiento de células individuales. Una limitación de este método es que este enfoque puede no ser adecuado en algunos entornos de enfermedad si se prevé que el cambio en la composición de las células inmunes y la e...

Los pulmones son un órgano barrera, expuestos al ambiente externo y, por lo tanto, están constantemente recibiendo desafíos inmunológicos como patógenos y alérgenos1. La activación de las células inmunes residentes en los pulmones y la infiltración de células inmunes de la periferia son necesarias para eliminar los patógenos del entorno pulmonar. Además, las células inmunes residentes en los pulmones mantienen la tolerancia a las bacterias comensales, lo que sugiere que estas células desempeñan un papel en la eliminación de patógenos y en el mantenimiento de la homeostasis1. Los macrófagos alveolares e intersticiales se encuentran entre las células inmunes centinela residentes en los pulmones que detectan patógenos a través de receptores de reconocimiento de patrones y eliminan estos patógenos por fagocitosis2. Las células dendríticas residentes en los pulmones unen la respuesta inmune innata y adaptativa a través de la presentación del antígeno3. Además, las células inmunes innatas locales activadas producen citoquinas y quimiocinas que amplifican la respuesta inflamatoria y estimulan la infiltración de células inmunes como monocitos, neutrófilos y linfocitos en los pulmones1. Se ha demostrado que el accidente cerebrovascular isquémico modifica la inmunidad sistémica y conduce a una mayor susceptibilidad a la infección pulmonar; Sin embargo, pocos estudios han evaluado el compartimento pulmonar después del accidente cerebrovascular isquémico, aunque algunos estudios lo han examinado durante condiciones inflamatorias 4,5,6,7,8,9. El objetivo de los métodos descritos en este documento es determinar simultáneamente la patología pulmonar, la composición de las células inmunes y los niveles de expresión de citoquinas y quimiocinas en los pulmones para evaluar las alteraciones en el compartimiento pulmonar y evaluar las posibles alteraciones en la respuesta inmune pulmonar después del ataque isquémico.
Aquí se describe un protocolo para obtener suspensiones de células individuales de los pulmones de los ratones para identificar 13 tipos de células inmunes. Este protocolo se basa en la digestión de tejidos con colagenasa D sin necesidad de un disociador tisular automatizado. Además, desarrollamos un protocolo para preparar homogeneizados tisulares que se pueden usar para determinar los niveles de expresión de 13 citoquinas o quimiocinas diferentes utilizando matrices de perlas multiplex basadas en citometría de flujo. Este protocolo se utilizó con éxito para investigar los efectos del accidente cerebrovascular isquémico en la inmunidad pulmonar y también se puede utilizar en otros modelos de enfermedad.

<table border="0" cellpadding="0" cellspacing="0" style="width:932px;" width="931">
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		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">B220-APC, clone RA3-6B2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103212</td>
			<td style="width:412px;">1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Beadbug 3 position bead homogenizer</td>
			<td style="width:129px;">Benchmark Scientific</td>
			<td style="width:101px;">D1030</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CCR2-BV421, clone SA203G11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">150605</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD103-BV421, clone 2E7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">121422</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11b-PE/Cy7, clone M1/70</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101216</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-PE/Cy7, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117318</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-Percp/Cy5.5, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117328</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD4-BV421, clone GK1.5</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100443</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD45-FITC, clone 30-F11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103108</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD64-APC, clone X54-5/7.1</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">139306</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD8-PE, clone 53-6.7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100708</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Collagenase D</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>11088882001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Conical screw cap tube</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">02-681-344</td>
			<td>Tube for tissue homogenization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">DNase I</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>10104159001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fc block CD16/32 antibody</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">genlteMACS dissociator</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Comparsion of lung digestion with or without mechanical dissociator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">gentleMACS C tubes</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td style="width:101px;">130-093-237</td>
			<td>Tube for tissue disscoiation with genlteMACS dissociator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Halt protease and phosphatase inhibitor cocktial</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">78442</td>
			<td>Component in the homogenization buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Laser doppler monitor</td>
			<td style="width:129px;">Moor</td>
			<td style="width:101px;">MOORVMS-LDF</td>
			<td>Blood flow monitoring during tMCAO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">LEGENDplex proinflammatory chemokine panel</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">740451</td>
			<td>Multiplex bead array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">LIVE/DEAD fixable near-IR stain</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">L34976</td>
			<td>Use for dead cell exclusion during flow cytometric analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6C-PE, clone HK1.4</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">128008</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6G-BV510, clone 1A8</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">127633</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MCAO suture L56 reusable 6-0 medium</td>
			<td style="width:129px;">Doccol</td>
			<td style="width:101px;">602356PK10Re</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">MHC II-BV510, clone M5/114.15.2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">107636</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">NK1.1-Percp/Cy5.5, clone PK136</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">108728</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Siglec F-PE, clone E50-2440</td>
			<td style="width:129px;">BD Biosciences</td>
			<td style="width:101px;">552126</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Silk suture thread, size 6/0</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td style="width:101px;">18020-60</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SomnoSuite anesthesia system</td>
			<td style="width:129px;">Kent Scientific</td>
			<td style="width:101px;">SS-01</td>
			<td>Mouse anaesthetization for tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">TCRb-BV510, clone H57-897</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">109234</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Zirconia/silica beads, 2.3 mm</td>
			<td style="width:129px;">Biospec</td>
			<td style="width:101px;">11079125z</td>
			<td>Beads for tissue homogenization</td>
		</tr>
	</tbody>
</table>

characterization of immune cells and proinflammatory mediators in the pulmonary environment

La expansión, activación y tráfico de células inmunes a los pulmones, que están controlados por la expresión de múltiples citoquinas y quimiocinas, pueden verse alterados por una lesión cerebral grave. Esto se evidencia por el hecho de que la neumonía es una causa importante de mortalidad en pacientes que han sufrido un accidente cerebrovascular isquémico. El objetivo de este protocolo es describir el uso del análisis citométrico de flujo multicolor para identificar 13 tipos de células inmunes en los pulmones de ratones, incluidos macrófagos alveolares, macrófagos intersticiales, células dendríticas (DC) CD103 + o CD11b +, CD plasmocitoides, eosinófilos, monocitos / células derivadas de monocitos, neutrófilos, células T y B derivadas de linfoides, células NK y células NKT, después de la inducción isquémica de accidente cerebrovascular por oclusión transitoria de la arteria cerebral media. Además, describimos la preparación de homogeneizados pulmonares utilizando un método de homogeneización de perlas, para determinar los niveles de expresión de 13 citoquinas o quimiocinas diferentes simultáneamente mediante matrices de perlas multiplex junto con análisis citométrico de flujo. Este protocolo también se puede utilizar para investigar la respuesta inmune pulmonar en otros entornos de enfermedades, como la enfermedad pulmonar infecciosa o la enfermedad alérgica.

Recientemente informamos que la inducción isquémica del accidente cerebrovascular en ratones altera la composición de las células inmunes de los pulmones11. Específicamente, la isquemia cerebral transitoria aumentó los porcentajes de macrófagos alveolares, neutrófilos y CD11b + DC, mientras que disminuyó los porcentajes de células T CD4 +, células T CD8 +, células B, células NK y eosinófilos en el compartimiento pulmonar. Además, la alteración celular correspondió a niveles signif...

Watch this Scientific Journal Video about Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment at JoVE.com

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Este protocolo describe el uso de la citometría de flujo para identificar los cambios en la composición de las células inmunitarias, el perfil de citoquinas y el perfil de quimiocinas en el entorno pulmonar después de la oclusión transitoria de la arteria cerebral media, un modelo murino de accidente cerebrovascular isquémico.

Caracterización de células inmunes y mediadores proinflamatorios en el ambiente pulmonar

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

This protocol can be used to investigate alterations in the pulmonary environment following ischemic stroke. This method allows for the identification of 13 lung immune cell types and 13 different cytokines and chemokines in the same mouse via flow cytometric analysis. This method has been utilized to assess alterations in the pulmonary environment following ischemic stroke.However, it can also serve to investigate changes during allergic response or infection. To perform whole body transcardial perfusion, confirm a lack of response to pedal reflect in an anesthetized mouse and secure the mouse to a surgical platform. Wet the fur with 70%ethanol to reduce fur distribution into the body cavity and make a vertical midline skin incision to expose the intact peritoneum stopping the incision as soon as the thoracic cavity has been reached.Use fine dissection scissors to make a midline incision of the peritoneum stopping at the thoracic cavity prior and use forceps to grasp the distal end of the sternum. Use the scissors to cut through the diaphragm without damaging the heart, lungs, or vasculature. When the underlying tissues are visible, make an incision through the rib cage and secure the sides of the ribs so that the heart and lungs are fully exposed.Carefully make a small incision in the right atrium near the aortic arch to serve as the outflow for the perfusion and quickly but gently insert a 25 gauge needle into the distal superior surface of the left ventricle. Once the needle is in place gently flush 10 milliliters of cold PBS into the heart. The tissue should begin to clear of blood.When the entire volume has been delivered, perfuse a second 10 milliliter aliquot of cold PBS until the liver exhibits a light brown appearance and the lungs have transitioned from a reddish pink to a mostly white in color. Using forceps and fine dissection scissors, carefully remove all of the surrounding tissues before separating the lung lobes and placing them into a Petri dish containing one to two milliliters of cold lung cell, medium on ice. To prepare the lung tissue for multiplex bead arrays add 200 microliters of cold homogenization buffer to a two milliliter tube containing three sterile 2.3 milliliters Zirconia/Silica beads, and weigh the lung lobe of interest.Transfer 50 to 100 milligrams of lung tissue to the pre-chilled tube of beads and homogenization buffer and homogenize the lobe using a bead based homogenizer at 4, 000 rotations per minute for at least two minutes. When the tissue has been completely homogenized, centrifuge the tube to sediment the tissue and transfer the super natant to a pre-chilled 1.5 milliliter micro tube on ice. For lung tissue dissociation carefully decant the lung cell medium from the remaining lung lobe and insert a 25 gauge needle into the tissue.Inject 200 to 250 microliter volumes of dissociation buffer into each lung lobe to inflate the lungs until each of the lobes have been injected two to three times. After a two minute incubation at room temperature use fine dissection scissors to mince the lungs into small pieces and transfer the tissue solution into 15 milliliter chronicle tube. Add six milliliters of additional dissociation buffer to the sample and vortex vigorously for one minute.Next, incubate the tissue pieces for 45 minutes at 37 degrees Celsius vortexing every seven to eight minutes. At the end of the incubation, vortex the sample vigorously for one minute and pass the sample through a 100 micron cell strainer to remove any residual undesired connective and interstitial tissue. Collect the tissue by centrifugation and resuspend to the pellet with five milliliters of cold lung medium.Centrifuge the sample again and resuspend the pellet in one milliliter of cold lung medium with gentle vortexing. Then, filter the cells through a 100 micron strainer before counting. To evaluate the immune cell populations within the isolated lung tissue, seed one to two times 10 to the sixth power cells antibody set into the appropriate number of wells with a 96 well round bottom plate and dispose the cells to the bottom of the plate by centrifugation.Add 25 microliters of the appropriate antibody cocktail in cold FACS buffer to each well and mix with gentle pipe petting. After 20 minutes, resuspend the cells in 100 microliters of cold FACS buffer per well and transfer the samples to individual five milliliter polystyrene round bottom tubes containing 150 microliters of additional FACS buffer per tube. Then, immediately analyze the samples on a flow cytometer according to standard flow cytometric analysis protocols.In this representative analysis, 13 different populations of immune cells were identified in the lungs using three sets of five to seven antibody combinations. Dead cells were excluded using a live dead stain and the different immune cell types were distinguished using the antibodies and markers listed in the table. Alveolar and interstitial macrophages, CD103 positive and CD11B positive dendritic cells and eosinophils were identified in set one.Pro-inflammatory monocytes and neutrophils were identified in set two and CD4 positive and CD8 positive T cells, B cells, plasmacytoid dendritic cells, NK cells, and NKT cells were identified in set three. The total number of viable cells percentage of CD45 positive cells and total number of CD45 positive cells obtained were similar between cell populations that were isolated as demonstrated and cell populations that were isolated using a commercially available tissue dissociator. In addition, the percentage of cell death among CD45 positive cells was less than 10%for both protocols, suggesting that the demonstrated protocol facilitates a high yield, high cell quality recovery without the aid of an automated tissue dissociator.Further, using a commercially available multiplex assay coupled with flow cytometric analysis allows the concentration of chemokine in each sample per milligram of tissue to be determined. The collagenase D concentration, incubation time and temperature used during the single cell isolation critically impacts the immune cell recovery.

characterization-immune-cells-proinflammatory-mediators-pulmonary

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Medición de la actividad calpaína en células fijadas y vivir por citometría de flujo

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Investigación

Inmunología e Infección

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Siete pasos para células estrelladas

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Aislamiento de células inmunes funcionales cardíacos

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagen intravital del timo de ratón utilizando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Diferenciar los roles funcionales de la expresión génica de las células inmunes y no inmunes en la colitis de ratón por Trasplante de Médula Ósea

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Ventricular derecha sistólica mediciones de presión en combinación con la cosecha de pulmón y muestras de tejido inmune en ratones

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Aislamiento de tejido adiposo células inmunes

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

El uso de ARN de interferencia para Investigar la respuesta inmune innata en macrófagos de ratón

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Aislamiento y caracterización de los neutrófilos con propiedades antitumorales

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

El aislamiento, caracterización y examen funcional de la red celular gingival Inmune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...