Vorremmo ringraziare il flusso Cytometry Core Facility presso The Pennsylvania State University Millennium scienza complessa.

<ol>
	<li>Davies, L. C., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages:+Then+and+Now.">Tissue-resident macrophages: Then and Now.</a> Immunology. 144 (4), 541-548 (2015).</li><li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (12), 840-846 (2006).</li><li>Van Gaal, L. F., Mertens, I. L., De Block, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+with+cardiovascular+disease.">Mechanisms linking obesity with cardiovascular disease.</a> Nature. 444 (12), 875-880 (2006).</li><li>Jung, U., Choi, M. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+and+Its+Metabolic+Complications:+The+Role+of+Adipokines+and+the+Relationship+between+Obesity,+Inflammation,+Insulin+Resistance,+Dyslipidemia+and+Nonalcoholic+Fatty+Liver+Disease.">Obesity and Its Metabolic Complications: The Role of Adipokines and the Relationship between Obesity, Inflammation, Insulin Resistance, Dyslipidemia and Nonalcoholic Fatty Liver Disease.</a> Int J Mol Sci. 15 (4), 6184-6223 (2014).</li><li>Emanuela, F., Grazia, M., Marco, D. R., Maria Paola, L., Giorgio, F., Marco, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+as+a+link+between+obesity+and+metabolic+syndrome.">Inflammation as a link between obesity and metabolic syndrome.</a> Nutr Metab. 2012, 1-7 (2012).</li><li>Vieira-Potter, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+macrophage+modulation+in+adipose+tissues.">Inflammation and macrophage modulation in adipose tissues.</a> Cell Microbiol. 16 (10), 1484-1492 (2014).</li><li>Chawla, A., Nguyen, K. D., Goh, Y. P. 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L., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+at+the+fork+in+the+road+to+health+or+disease.">Macrophages at the fork in the road to health or disease.</a> Front Immunol. 6 (2), 1-6 (2015).</li><li>Italiani, P., Boraschi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+monocytes+to+M1/M2+macrophages:+Phenotypical+vs.+functional+differentiation.">From monocytes to M1/M2 macrophages: Phenotypical vs. functional differentiation.</a> Front Immunol. 5 (8), 1-22 (2014).</li><li>Guilherme, A., Virbasius, J. V., Vishwajeet, P., Czech, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+liver+disease+of+obesity+and+role+of+adipose+tissue+in+the+pathogenesis+of+nonalcoholic+fatty+liver+disease.">Metabolic liver disease of obesity and role of adipose tissue in the pathogenesis of nonalcoholic fatty liver disease.</a> World J Gastroenterol. 13 (26), 3540-3553 (2007).</li><li>Bedossa, P., Paradis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+extracellular+matrix+in+health+and+disease.">Liver extracellular matrix in health and disease.</a> J Pathol. 200 (4), 504-515 (2003).</li><li>Tilg, H., Moschen, A. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity,+atherosclerosis+and+the+vascular+endothelium:+mechanisms+of+reduced+nitric+oxide+bioavailability+in+obese+humans.">Obesity, atherosclerosis and the vascular endothelium: mechanisms of reduced nitric oxide bioavailability in obese humans.</a> Int J Obesity. 26 (12), 754-764 (2002).</li><li>Mestas, J., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Endothelial+Cell+Interactions+in+the+Development+of+Atherosclerosis.">Monocyte-Endothelial Cell Interactions in the Development of Atherosclerosis.</a> Trends Cardiovasc Med. 18 (6), 228-232 (2008).</li><li>Wynn, T. A., Chawla, A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+biology+in+development,+homeostasis+and+disease.">Macrophage biology in development, homeostasis and disease.</a> Nature. 496 (7446), 445-455 (2013).</li><li>Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages.">Tissue-resident macrophages.</a> Nat Rev Immunol. 14 (10), 986-995 (2013).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> J Vis Exp. (41), e1546 (2010).</li><li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+Identification,+and+Excision+of+Murine+Adipose+Depots.">Localization, Identification, and Excision of Murine Adipose Depots.</a> J Vis Exp. (94), e52174 (2014).</li><li>Butcher, M. J., Herre, M., Ley, K., Galkina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+immune+cells+within+murine+aortas.">Flow cytometry analysis of immune cells within murine aortas.</a> J Vis Exp. (53), e2848 (2011).</li><li>Salamone, M., Saladino, S., Pampalone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Dissociation+and+Primary+Cells+Isolation+Using+Recombinant+Collagenases+Class+I+and+II.">Tissue Dissociation and Primary Cells Isolation Using Recombinant Collagenases Class I and II.</a> Chemical Engineering Transactions. 38, 247-252 (2014).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47 (4), 1394-1400 (2008).</li><li>Fain, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Free+Brown+and+White+Fat+Cells.">Isolation of Free Brown and White Fat Cells.</a> Diabetologia. 375 (1964), 555-561 (1968).</li><li>Seglen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Method Cell Biol. 13, 29-83 (1976).</li><li>Yu, S., Allen, J. N., 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+Ron+Receptor+Tyrosine+Kinase+Regulates+Macrophage+Heterogeneity+and+Plays+a+Protective+Role+in+Diet-Induced+Obesity,+Atherosclerosis,+and+Hepatosteatosis.">The Ron Receptor Tyrosine Kinase Regulates Macrophage Heterogeneity and Plays a Protective Role in Diet-Induced Obesity, Atherosclerosis, and Hepatosteatosis.</a> J Immunol. 197 (1), 256-265 (2016).</li><li>Yu, Y. R. A., O'Koren, E. 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=A+protocol+for+the+comprehensive+flow+cytometric+analysis+of+immune+cells+in+normal+and+inflamed+murine+non-lymphoid+tissues.">A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues.</a> PLoS ONE. 11 (3), 1-23 (2016).</li><li>Zizzo, G., Hilliard, B. A., Monestier, M., Cohen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+clearance+of+early+apoptotic+cells+by+human+macrophages+requires+&amp;#34;M2c&amp;#34;+polarization+and+MerTK+induction.">Efficient clearance of early apoptotic cells by human macrophages requires &amp;#34;M2c&amp;#34; polarization and MerTK induction.</a> J Immunol. 100 (2), 130-134 (2012).</li><li>Jablonski, K. A., Amici, S. A., 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=Novel+markers+to+delineate+murine+M1+and+M2+macrophages.">Novel markers to delineate murine M1 and M2 macrophages.</a> PLoS ONE. 10 (12), 5-11 (2015).</li><li>R&#337;szer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+Mysterious+M2+Macrophage+through+Activation+Markers+and+Effector+Mechanisms.">Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms.</a> Mediators of Inflamm. 2015, 1-16 (2015).</li><li>Cho, K. W., Morris, D. L., Lumeng, C. 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Gli autori non hanno alcun conflitto di interessi di divulgare.

Modelli di malattia metabolica indotta da dieta che imitano co-morbidità, quali aterosclerosi, semplice steatosi e steatoepatite e diabete di tipo 2 sono ampiamente utilizzati per comprendere meglio i meccanismi molecolari della progressione di malattia. Dipendente dalla digestione della collagenosi viene spesso utilizzata per dissociare tessuti per liberare le cellule dalla matrice extracellulare (ECM)16,27. Enzimi quali la collagenasi perturbare collagene che ...

Modelli murini sono stati ampiamente utilizzati per studiare la dinamica delle malattie umane. Adeguato isolamento delle cellule residenti del tessuto da topi in uno stato malato può fornire una piattaforma per comprendere i contributi molecolari e cellulari alla patogenesi di una malattia1. Un disordine che è di fondamentale importanza è l'obesità. L'incidenza dell'obesità continua ad aumentare in tutto il mondo in parallelo con l'insulino-resistenza e di tipo 2 mellitus del diabete, malattia cardiovascolare e steatosi epatica2,3. Eccessivo consumo di nutrienti è ulteriormente inclinato da riduzione dell'attività fisica innescando segnali alterati provenienti dal tessuto adiposo, che può alterare l'ambiente cellulare degli altri tessuti periferici quali l'aorta e fegato4. Tale perturbazione dell'omeostasi metabolica si traduce in un di infiammazione sistemica cronica di basso grado5.
Classica attivazione dei macrofagi residenti per l'aorta e fegato, nonché il loro reclutamento al tessuto adiposo bianco (WAT) ha dimostrato di non solo avviare il dysregulation di segnali metabolici, ma anche sostenere l'infiammazione6,7. L'eterogeneità fenotipica e funzionale dei macrofagi è fortemente associato con la patogenesi dell'obesità correlate a co-morbidità7. La plasticità dinamica nella polarizzazione del macrofago consente queste cellule ad esporre una gamma di fenotipi attivati che coordinano la progressione e la risoluzione di infiammazione8. Mentre i macrofagi (M1) classicamente attivati sono implicati nella propagazione dell'infiammazione, i macrofagi (M2) in alternativa attivati sono stati associati con risoluzione e tessuto riparazione9,10.
Come il corpo subisce stress metabolico, tessuto adiposo bianco si accumula in modo anomalo. Il tessuto adiposo espanso attira e trattiene le cellule infiammatorie che profondo alterano la normale funzione degli adipociti per promuovere l'insulino-resistenza, iperglicemia e diabete mellito di tipo 2 in ultima analisi, l'insulino-resistenza o iperglicemia11, 12. In parallelo, tessuto adiposo bianco rimodella in risposta a segnali infiammatori rilasciato da infiltrato classicamente attivati (M1) tessuto adiposo macrofagi (ATMs)13,14. Questo organo multi-cellulare esercita una cascata di segnali che deraglia la normale funzione di altri organi del corpo come l'aorta e fegato4.
Il fegato è un concentrato di metabolico che si adatta in risposta a stimoli provenienti da vicine dysregulated WAT15. Epatici macrofagi o cellule di Kupffer, in risposta a cambiamenti metabolici, secernono citochine infiammatorie che trasformano entrambi parenchimatica e fenotipo delle cellule non parenchimali e promuovono il rimodellamento tissutale. Accumulo di lipidi epatici, infiammazione, depositi eccessivi di matrice extracellulare, necrosi e funzione eventuale perdita segue gli insulti infiammatori contribuendo ad ampio spettro di danni al fegato associati con epatopatia steatosica non alcolica 16,17,18.
In parallelo al compromesso WAT e funzione epatica, grandi arterie si accumulano i lipidi all'interno della parete arteriosa come il corpo subisce lo stress metabolico cronico19. Accumulazione del lipido arteriosa innesca la secrezione di chemochine dalle cellule endoteliali attivate e successiva assunzione di monociti20. Una volta reclutate, monociti proliferano, differenziano, ingeriscono lipoproteine e diventano cellule della gomma piuma. Atherogenesis è avviata e sostenuta da attività pro-infiammatoria di reclutati e macrofagi lipido-carichi residente in tessuto. Soccombere ai segnali extracellulari ed intracellulari stress inoltrati in questo microambiente atherogenic, questi macrofagi quindi impegnano in una cascata di segnalazione di apoptotic. Come queste cellule schiumose muoiono, essi contribuiscono loro contenuto lipidico riempito al nucleo necrotico della lesione, che poi conduce alla rottura della placca, infarto miocardico e ictus.
Collettivamente, l'eterogeneità fenotipica del macrofago orchestra in parte l'obesità indotta da cambiamenti infiammatori osservati in tessuti dysregulated come WAT, fegato e dell'aorta8,21. Caratterizzazione di reclutati e macrofagi residenti tessuto potrebbero fornire la comprensione nei potenziali bersagli molecolari che manipolano macrofago fenotipo1. Per caratterizzare in modo efficace i macrofagi dai tessuti infiammati obesità-indotta, una sospensione di singola cellula può essere ottenuta tramite digestione enzimatica. Tali protocolli di dissociazione devono essere efficace nel sufficientemente degradante tessuto connettivo riducendo la morte delle cellule immuni e fornendo resa cellulare ottimale. La miscela di enzimi dipende dal tipo di tessuto e suo strutturale compongono. Tessuti resistenti, come l'aorta richiede più forte attività enzimatica, rispetto al fegato e WAT, per raggiungere la dissociazione dei tessuti. Dalla sospensione unicellulare, macrofagi residenti del tessuto possono essere inequivocabilmente caratterizzati o isolati per ulteriore analisi, a valle quali profiling trascrizionale.
Qui un protocollo di tessuto-specifico è descritto che utilizza la digestione della collagenosi-dipendente del tessuto e citometria a flusso policromatici efficacemente isolare e caratterizzare i macrofagi residenti in tessuto ottenuti da obesità dieta tradizionale ha indotto, aterosclerosi, semplice steatosi e steatoepatite modelli murini. Simultanea di colorazione degli indicatori della superficie delle cellule con anticorpi contro del leucocita-(CD45 e/o CD11b) e del macrofago - antigeni specifici (F4/80) è spesso utilizzato per identificare il macrofago popolazioni22. Fluorescenza-attivato delle cellule ordinano (FACS) sono una strategia potente utilizzata per ordinare queste popolazioni identificate a elevata purezza. La popolazione ordinata quindi possa essere valutata per profili di gene specifico fenotipo mediante analisi molecolare a valle (ad esempio, reazione a catena della polimerasi quantitativa real time)23. Sebbene la citometria a flusso standard e flusso cytometry-base cella ordinamento sono strumenti potenti nel distinguere i macrofagi all'interno di una sospensione cellulare notevolmente eterogenei, i protocolli ex devono essere ottimizzati per garantire successo uscita. In questo studio, protocolli che efficacemente isolare e caratterizzano i macrofagi specifico tessuto vitale sono descritti; ancora più importante, questo studio fornisce approfondimenti cruciali di questioni tecniche che spesso sorgono, così come le strategie proattive e Trouble-Shooting per prevenire e/o risolverli.

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">26G x 5/8 in Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305115</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">23G x 0.75 in needle x 12 in. tubing Blood Collection Set</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">367297</td>
			<td style="width:349px;">Used for cannulation of subhepatic IVC during liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">21G 1 1/2 in. Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305156</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL syringe with rubber stops</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">10mL Syringes</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309604</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL Syringe</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">F4/80 PE</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">123110</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11c PE/Cy7</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">117318</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11b PE/Cy5</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:119px;">15-0112-81</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-mouse CD16/32 Fc Block&#160;</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">101320</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD45 Pacific Blue</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">103126</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE Rat IgG2a</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400508</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy7 Armenian Hamster IgG</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400922</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy5 Rat IgG2b</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400610</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Pacific Blue Rat IgG2c</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400717</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">15-017-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1X Phosphate Buffered Saline (PBS)</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">70 micron cell strainers</td>
			<td style="width:151px;">Corning, Inc.</td>
			<td style="width:119px;">352350</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">&#160;1.7 mL microcentrifuge tube&#160;</td>
			<td style="width:151px;">Denville</td>
			<td style="width:119px;">C2170</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Paraformaldehyde Aqueous Solution -16X</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">CAS #30525-89-4</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Micro Dissecting Scissors, 3.5 inch, Straight, 23mm, Sharp&#160;</td>
			<td>Stoelting</td>
			<td>52132-10P</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Forceps, 4in, full curve, 0.8mm</td>
			<td>Stoelting</td>
			<td>52102-37P&#160;</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Spring dissection scissors &#8211; 3 mm Cutting edge&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">15000-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Curved 0.07 x 0.04 mm Tip Forceps&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">11297-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hemostatic Forceps (Curved)</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">13021-12</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Heparin Sodium Salt</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">9041-08-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">35mm Cell Culture/Petri Dishes</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">12-565-90</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Polystyrene Petri Dishes (10 cm) w/lid</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">08-757-100D</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">15mL Conical Centrifuge Tubes (Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50mL Conical Centrifuge Tubes&#160;(Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">5mL Round-Bottom Polystyrene Tubes&#160;</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-5</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:151px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td>
			<td style="width:151px;">Millipore</td>
			<td style="width:119px;">EX0285-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Bovine Serum Albumin</td>
			<td style="width:151px;">Rockland</td>
			<td style="width:119px;">BSA-50</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type II</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenasse Type XI</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hyaluronidase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3506</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">DNAse</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0130</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">NaOH</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">1310-73-2&#160;</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:321px;">CaCl2</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">500mL beaker&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">02-540M</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">4 cm Hemostatic clamp</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52120-40</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Toothed forceps</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52104-33P</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50 micron Disposable filters</td>
			<td style="width:151px;">Systemex</td>
			<td style="width:119px;">04-0042-2317</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Collagenase Type IV</td>
			<td style="width:151px;">ThermoFischer Scientific</td>
			<td style="width:119px;">17104019</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Ammonium Chloride Potassium (ACK)</td>
			<td style="width:151px;">ThermoFischer Scientific&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Razors (0.22 mm (0.009&#34;))</td>
			<td style="width:151px;">VWR International</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Texas Red Live/Dead stain</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"></td>
			<td style="width:349px;">Red viability stain (in Figure 1A)</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

L'obesità favorisce uno stato infiammatorio cronico che è in gran parte mediato dai macrofagi residenti in tessuto così come macrofagi monocito-derivati. L'obesità indotta da dieta (DIO) è un modello importante nello studio del ruolo di eterogeneità dei macrofagi; Tuttavia, gli isolamenti del macrofago adeguata sono difficili da acquisire da tessuti infiammati. In questo protocollo, vengono delineati i passaggi di isolamento e le necessarie linee guida sulla risoluzione dei problemi derivate dai nostri studi per l'ottenimento di una popolazione adatta dei macrofagi residenti in tessuto da topi dopo 18 settimane di grassi (HFD) o alto-grasso/colesterolo ( Intervento di dieta HFHCD). Questo protocollo si concentra su tre hallmark tessuti studiati nell'obesità e aterosclerosi compreso il fegato, tessuto adiposo bianco (WAT) e l'aorta. Evidenziamo l'utilizzo come dualistica di flusso cytometry può raggiungere una nuova dimensione di isolamento e caratterizzazione dei macrofagi residenti in tessuto. Una sezione fondamentale del presente protocollo risolve la complessità sottostante digestioni enzimatiche tessuto-specifici e isolamento del macrofago e successiva anticorpo cellula-superficie che macchia per analisi cytometric di flusso. Questo protocollo consente di risolvere complessità esistente alla base fluorescente-attivata delle cellule ordinano (FACS) e presenta chiarimenti a queste complessità in modo da ottenere la caratterizzazione di vasta gamma da popolazioni cellulari adeguatamente ordinato. Metodi di arricchimento alternativo sono inclusi per l'ordinamento di cellule, quali il fegato denso, consentendo per la gestione di tempo e flessibilità quando si lavora con FACS. In breve, questo protocollo aiuti al ricercatore di valutare l'eterogeneità dei macrofagi da una moltitudine di tessuti infiammati in un dato studio e fornisce penetranti suggerimenti sulla risoluzione dei problemi che hanno avuto successo per la caratterizzazione e isolamento cellulare favorevole delle cellule immuni in DIO-ha mediato l'infiammazione.

Quando si utilizza dell'apolipoproteina E (ApoE KO) C57BL/6 (BL6) topi mantenuti su una dieta di alto colesterolo alto grasso (HCHFD o HCD) per 18 settimane, 1 x 104 - 2 x 104 CD45++ F4/80 macrofagi aortici possono essere isolati, quando i due campioni sono in pool. Fegati sezionati da topi KO per ApoE HFHCD-Federazione, prodotto maggiore di cellule di Kupffer5 ordinato 5 x 10 (che dipende dal tempo di ordinamento disponibile). Quando...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Questo protocollo consente un ricercatore di isolare e caratterizzare residente in tessuto hallmark macrofagi in vari tessuti infiammati estratte dai modelli indotta da dieta di disordini metabolici.

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

isolation-characterization-purification-macrophages-from-tissues

Research

JoVE Journal

Immunology and Infection

25.1K Views.  The Pennsylvania State University. Questo protocollo consente un ricercatore di isolare e caratterizzare residente in tessuto hallmark macrofagi in vari tessuti infiammati estratte dai modelli indotta da dieta di disordini metabolici.

Video: Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Isolamento e caratterizzazione di cellule dendritiche ei macrofagi dall&#39;intestino mouse

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Ricerca

Immunologia e infezioni

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomica Profiling dei macrofagi da 2D elettroforesi

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un metodo ottimizzato per l&#39;isolamento ed espansione invariante Natural Killer cellule T da Spleen mouse

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

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.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

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

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Caratterizzazione funzionale dei macrofagi regolatori che inibiscono l&#39;immunità reattiva dell&#39;inferta

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolamento e caratterizzazione di vescicole extracellulari da adulto Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Analisi sperimentale di apoptotic thymocyte engulfment da macrofagi

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...