This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG14593, MFAG13235), the Fondazione Cariplo (Grant 2010-0678 and NANOVAC) and the Fondazione Regionale per la Ricerca Biomedica.

Studienzulassung: Die experimentellen Protokolle wurden vom italienischen Ministerium für Gesundheit genehmigt (Rom, Italien) nach dem Decreto legislativo 27 novembre 1992, n. 116 &quot;attuazione della Direttiva n. 86/609 / EWG in materia di Protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici.&quot; 1. Gewinnung Hautproben <ol><li> Euthanize die Maus durch Genickbruch. </li><li> Ohr: <ol><li> Schneiden Sie den unbehaarten Teil der Ohren der Maus aus. </li><li> Separate dorsalen und ventralen Seite durch sie auseinander mit einer Pinzette herausziehen. Entfernen Sie alle verbleibenden Knorpeln durch sanft den inneren Teil der Ohren mit einer Pinzette Schaben. </li></ol></li><li> Trunk Haut: <ol><li> Rasieren Sie die ganze Haut des Tieres sowohl mit einem elektrischen Rasierer und mit zarten Enthaarungslotion. </li><li> Schneiden Sie die gewünschte Hautprobe (von 2 cm 2 bis zur gesamten Tierhaut) aus. <ol><li> Wenn die gesamte Rücken abschneidet und ventrale Mäusehaut, einen vertikalen Schnitt in der Mitte der Maus entlang der Grenzen der rasierten Bereichen rund um die Maus Körper zurück geschnitten und dann machen. </li><li> Vorsichtig trennen die Haut aus dem Peritoneum und Rückenmuskulatur, immer noch mit der Pinzette die Hautprobe zu halten, während die Hautprobe mit geschlossenen runden kantigen Spitzen einer chirurgischen Schere oder einem Skalpell trennt. </li></ol></li><li> Bereiten Sie eine 100 mm Zellkulturplatte mit 10 ml eiskaltem PBS enthält. </li><li> Beseitigen von subkutanem Fett durch Eintauchen der Hautprobe in PBS in der Platte, hergestellt in Schritt 1.3.3 und Abkratzen der Hautprobe mit einer Pinzette. </li></ol></li><li> Schwanz: <ol><li> Schneiden Sie den Schwanz der Maus ab. </li><li> Machen Sie einen vertikalen Schnitt auf den Schwanz mit einer chirurgischen Schere oder chirurgische Skalpell aus der Schwanzbasis beginnt und an der Spitze des Schwanzes zu erreichen. </li><li> Verwenden einer Pinzette, um die Haut vom Schwanz zu lösen. Besorgen Sie sich die Kante des Schnitts an der Basis des Schwanzes und ziehen Sie vorsichtig die Spitze zu, während die HalteKörper des Schwanzes mit einer Pinzette. </li><li> Entfernen Sie überschüssiges Fett durch die Hautprobe sanft kratzen. </li></ol></li><li> Wegelagerer <ol><li> Schneiden Sie die gesamte Maus Fuß mit chirurgischen Schere. Vermeiden jede behaarter Haut zu schneiden. </li><li> Schneiden Sie die Haut des Fußes in Längsrichtung vom Knöchel bis zum Beginn der Ziffern. </li><li> Halten Sie die Knöchel mit einer Pinzette oder mit der Hand, während die Haut mit einer Pinzette greifen und ziehen Sie die Haut in Richtung auf die Ziffern, als ob, die ein Handschuh aus. </li></ol></li></ol> 2. Verdauung <ol><li> Bereiten Sie die Verdauung Cocktail aus der Stammlösung (hergestellt wie in der Materialliste) mit folgendem Rezept: Enzymmischung (siehe Materialliste für Spezifikationen) 300 ug / ml, DNAse1 50 U / ml. Man verdünnt in RPMI 5% FBS. </li><li> Hacken Hautproben mit einer Schere in kleine Stücke in eine 35 mm Petrischale mit 2 ml Digestion Cocktail (Für Stamm Haut: 2 ml Verdauung Cocktail alle 2 cm 2 der Haut, bis zu 3 ml insgesamt). </li><li>für 90 min bei 37 ° C inkubieren. Schütteln Sie die Petrischale 1-2 mal während der Inkubation. </li><li> Gießen verdaute Hautproben auf einem 70 &amp; mgr; m Zellsieb in 66 mm Petrischale mit 1 ml RPMI1640 5% FBS. </li><li> Waschen Sie das Sieb mit 5 ml RPMI 1640 5% FBS. </li><li> Drücken Sie die Probe durch die Zelle Sieb mit einem Spritzenkolben. </li><li> Flush Kolben, Petrischale und Sieb mit 20 ml RPMI1640 5% FBS, Transfer in 50-ml-Tube. </li></ol> 3. Antikörper und Labeling <ol><li> Waschen der erhaltenen Suspension zweimal mit 5 ml PBS, das 1% FBS (oder BSA). </li><li> Beschriften Sie die erhaltene Zellsuspension mit den gewünschten Antikörper. <ol><li> Resuspendieren der Proben in 100 ul / Probe einer Mischung enthaltend 2 &amp; mgr; g / ml aCD16 / CD32 (Klon 2.4G2), verdünnt in PBS 1% FBS. </li><li> 15 min Inkubieren bei 4 ° C, um nicht-spezifische Bindung der gewünschten Antikörper via Fc-Rezeptoren zu blockieren. </li><li> Fügen Sie die angegebenen Antikörper in der Tabelle1. <ol><li> Bereiten einer Mischung die gewünschten Antikörper in 100 &amp; mgr; l / Probe von PBS 1% FBS in einer Konzentration von 4 ug / ml verdünnt. </li><li> Füge 100 &amp; mgr; l des in Schritt hergestellten Mischung 3.2.3.1 zu jeder Probe, so dass in dem Rohr, wird es in einer Endkonzentration von 2 &amp; mgr; g / ml mit einem Volumen von 200 &amp; mgr; l der Antikörpermischung sein. </li></ol></li><li> Inkubieren für 30 min bei 4 ° C und die Abdeckung von Licht. </li></ol></li><li> Waschen mit 1 ml PBS, das 1% FBS, Zentrifuge für 5 Minuten bei 400 · g, den Überstand verwerfen und resuspendieren in 300 ul PBS, das 1% FBS. </li><li> Hinzufügen DAPI in einer Endkonzentration von 2 &amp; mgr; g / ml und Inkubation bei 4 ° C, geschützt vor Licht für 10 min. </li><li> Analysieren Sie die Proben durch FACS erhalten. <ol><li> Um Leukozyten zu isolieren, verwenden Sie die folgende Gating-Strategie: <ol><li> Zuerst beseitigen alle toten Zellen durch auf der DAPI- Bevölkerung Gating. </li><li> Wählen Sie Singuletts im FSC-A gegen FSC W Plot , wie in Abbildung 1 angegeben </strong&gt;. </li><li> Tor die darauf basierenden Zellen nach Größe und Granularität typisch für die Leukozyten von Interesse. </li><li> Wählen Sie CD45 + Zellen in der FSC-A CD45 Plot wie in Abbildung 1 dargestellt. </li></ol></li><li> Unterscheiden die verschiedenen Populationen von Leukozyten durch die Expression von Oberflächenmarkern, wie folgt: <ol><li> Identifizieren DCs als CD11c + MHC II + Zellen. Diese können später für die Expression von CD207 analysiert werden, um zu unterscheiden, Langerhans&#39;schen Zellen und CD207 + Dermal DCs von CD207- Dermal DCs. </li><li> Identifizieren Makrophagen als CD64 + F4 / 80 + Zellen. </li><li> Identifizieren B-Zellen als CD19 + MHCII + Zellen. </li><li> Identifizieren T-Zellen als CD8 + oder CD4 + Zellen. </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Wir haben ein Verfahren zur Herstellung von Einzelzellsuspensionen aus verschiedenen Maushautregionen beschrieben. Verfahren nach Verdauung wir angenommen haben, verursacht nicht nur hohe Ausbeuten, sondern bewahrt auch die Expression von Oberflächenmarkern, die für die nachfolgende FACS-Analyse von grundlegender Bedeutung ist. Die Verwendung eines Cocktails von Kollagenase I und II und Thermolysin, garantiert minimale Charge zu Charge unterschiedliche Enzymaktivität macht dieses Verfahren sehr gut reproduzierbar. Andere veröffentlichte Methoden beruhen auf verschiedenen Enzymcocktails aber in unseren Händen geben sie niedrigere Erträge und, was noch wichtiger ist, sind weniger reproduzierbar. Unser Verfahren wird jedoch nicht zwischen Zellen bevölkern die Epidermis und die Dermis Zellen füllen. Da in vielen Fällen könnte es nützlich sein , zwischen diesen beiden verschiedenen Kompartimenten zu unterscheiden, richten wir den Leser auf diesen früheren Arbeiten , die solche Verfahren verwenden. 15 Nach unserer Erfahrung Hautprobes aus dem Ohr und Schwanz sind leichter auf den Stamm Haut verdauen, als wo die subkutane Fettschicht, um gründlich abgekratzt werden muss, die Dermis zugänglich für die Enzym-Cocktail zu machen. Die Entfettung Schritt, obwohl sowie das Schneiden sollte schnell und sanft ausgeführt werden, um die Lebensfähigkeit der Zellen aufrecht zu erhalten. Eine verlängerte oder zu gründlich Entfettungsschritt kann zu Schäden an der Immunzellen mit Wohnsitz in der Haut führen und daher in viel geringeren Ausbeute führen und verminderte Lebensfähigkeit der gewonnenen Zellen. Es sollte auch beachtet werden, dass die Integrität der Oberflächenmarker zu erhalten, sollte die Verdauung von Reduktionsmittel wie β-Mercaptoethanol in einem Medium frei durchgeführt werden. Um unerwünschte Schäden zu vermeiden Marker zur Oberfläche, sollte die Inkubationszeit kürzer als wie möglich gehalten werden, in jedem Fall nicht länger als 90 min verlängert. Besondere Sorgfalt sollte auch werden, genommen, wenn die verdaute Hautproben in die Zelle GießenSieb. Es ist wichtig, gründlich verdaut Haut, um waschen die befreiten Zellen vor der Zerschlagung der verdauten Probe mit dem Spritzenkolben weg zu waschen. Mit diesem Schritt wird die Hautprobe aus den befreiten Immunzellen gewaschen, die dann nicht mehr körperlicher Belastung unterziehen. Die Zerschlagung sollte auch vorsichtig erfolgen, um keine Zellen und sowohl die Zelle Sieb und der Spritzenkolben zu töten sollte gründlich in der nachfolgenden Waschschritt gewaschen werden. Haut Verdauung kann auch in einem 50 ml Falcon in einem Wasserbad bei 37 ° C durchgeführt werden. In diesem Fall kann die Haut vorher gehackt werden und dann in den Falcon-Röhrchen. Beachten Sie, dass die Hautproben immer in Verdauungspuffer während der Inkubation und dass Hörmuscheln halten Sie sich an den Seiten des Falcon-Röhrchen getaucht werden sollte. Wenn Falcon-Röhrchen verwenden, daher sicherstellen, dass die Stücke gehackte Haut Aufenthalt in der Digestionsmedium untergetaucht. Dieses Verfahren kann in ma nützlich seinny verschiedenen experimentellen Bedingungen, vor allem, wenn nicht-entzündete Haut studieren. Während einer Entzündung, Vasodilatation und Gewebeschwellung lösen die Struktur des Gewebes und Immunzellen aus dem Blut massiv auf die Haut rekrutiert werden. Die Effizienz der Zellgewinnung ist somit viel höher. In homöostatischen Bedingungen ein effektiver Weg, den tight Kollagenmatrix der Dermis ist, in der Tat erforderlich, um zu verdauen, um die Immunzelle bildet das komplexe Netzwerk Bestücken der Haut wiederherzustellen. Der Leser könnte auch in einer umfassenden Überprüfung interessiert sein, die den Ursprung und Phänotyp verschiedener dendritische Zellen und Makrophagen bevölkern die Haut in den beiden homeostatic und entzündlichen Erkrankungen abdeckt. 16 In verschiedenen Versuchseinstellungen können verschiedene Abschnitte der Mäusehaut behandelt werden. Zum Beispiel in Hauttransplantationsmodellen könnte ein Teil des Schwanzes auf dem Kofferraum eines Aufnahme Tier transplantiert werden; wenn studying Ödembildung oder Immunisierungsverfahren, Stimuli werden häufig auf den Fußballen von Mäusen injiziert, während das Ohr häufig T-Zellenspeicher mit DTH-Tests zur Beurteilung verwendet wird. Mit unserer Methode können wir effizient Haut resident Immunzellen von verschiedenen Standorten aus, dass unser Verfahren nützlich für die experimentelle Einstellung der Wahl extrahieren.

The skin is one of the largest organs of the body and its large surface is continuously exposed to the external environment. Therefore the skin has to protect the organism from potential threats in order to maintain homeostasis, both by physical means, and by providing active protection toward potential pathogen entry. In a similar way to the gut and lung mucosa, the skin homes a variety of immune cells that continuously interact with the epithelium in order to maintain immune surveillance. This complex system that involves both immune and non-immune cells of the skin has been acknowledged since the early days of immunology, when in 1978 the term "Skin-associated lymphoid tissue" (SALT) 1 was first coined to describe such complexity. 
The large number of immune cells residing in the skin helps the organism not only to fight potential invasions, but also to orchestrate wound healing and to maintain tolerance toward self-antigens and the skin microbiota 2,3. 
Amongst skin resident immune cells, Dendritic Cells (DCs) have a crucial role in shaping immune responses and maintaining homeostasis 4. Dermal DCs and Langerhans cells readily respond to pathogen invasions and, upon migration to lymph nodes activate T cells and induce the expression of skin homing receptors on the newly generated effector T cells 5. DCs also have a fundamental role in regulating skin homeostasis. DCs migrating from the dermis to the lymph nodes in homeostatic conditions transport skin sequestered antigens with the purpose of inducing T cell tolerance mostly through the differentiation of regulatory T cells (Tregs) specific for skin antigens 6-8. 
T cells represent the most abundant population of immune cells in the skin. Not only do they infiltrate the skin during an infection but they also represent a stable population among skin resident lymphocytes 9,10. Both CD8+ and CD4+ resident memory (rm)T cells reside in the skin and, following an infection, can respond long before effector T cells are recruited from the blood. In the skin, also resides a population of tissue resident Tregs capable of maintaining tolerance toward skin antigens and tissue homeostasis. These cells are rapidly and potently activated after exposure to their cognate antigen and have therefore been defined as memory Tregs 11,12. 
Along with DCs and T cells, many innate immune cells, such as NK cells, gamma delta T cells, group 2 ILCs 13, Mast Cells and Macrophages, populate the skin and contribute to host protection. To analyze such a complex environment, it is mandatory to obtain single-cell suspensions from skin specimens with high efficiency while at the same time preserving the expression of surface markers for flow cytofluorimetric analysis or sorting. 
Murine skin presents an outer epidermal layer, constituted mainly of keratinocytes, Langerhans cells and dendritic epidermal T cells; and the dermal layer beneath. The dermis homes most of the immune cells and is made by an extracellular matrix in which collagen fibers are the most abundant, especially collagen-I and collagen-IV. Unlike human skin, mouse skin is covered in fur, and thus more populated with hair follicles. It is, also, much thinner than human skin and contains a muscular layer, the panniculus carnosus, which helps wound healing 14. These characteristics render it more difficult to efficiently disrupt the collagen net of the dermis in order to get immune cells out.
In this paper we provide a suitable method for digesting the extracellular matrix that relies on a cocktail of high purity collagenase 1 and 2 and thermolysin. Moreover, since the analysis of skin from different regions requires different experimental procedures, we will show different methods to efficiently obtain skin samples from the footpad, tail, ear and trunk. We will then label the samples in order to evaluate the presence of DCs (MHCII+CD11c+CD45+) Macrophages (CD11b+F4/80+CD45+), CD8+, and CD4+ T cells.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Reagents</td>
			<td style="width:88px;"></td>
			<td style="width:155px;"></td>
			<td style="width:276px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:203px;">Supplemented RPMI 1640 medium&#160;</td>
			<td></td>
			<td></td>
			<td style="width:276px;">RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Liberase TM (Enzyme Mix)</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td style="width:276px;">resuspend [2.5mg/ml] in dH20 store aliquots at -20&#176;C&#160;</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Dnase I</td>
			<td>Sigma&#160;</td>
			<td>D4263-1VL</td>
			<td style="width:276px;">resuspend in RPMI 1640 w/o serum [1000u/ml]</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">35mm petri dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">66mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">75um Cell Strainers</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Clone</td>
			<td>Label</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD45.2</td>
			<td></td>
			<td>104</td>
			<td>PE-Cy5.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD11c</td>
			<td></td>
			<td>N418</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CD11b</td>
			<td></td>
			<td>M1/70</td>
			<td>FITC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">F4/80</td>
			<td></td>
			<td>A 3-1</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD4</td>
			<td></td>
			<td>Gk1.5</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD8</td>
			<td></td>
			<td>53-6.7</td>
			<td>PE</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD64</td>
			<td></td>
			<td>X54-5/7.1</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD207</td>
			<td></td>
			<td>4C7</td>
			<td>APC</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Digestion cocktail</td>
			<td></td>
			<td></td>
			<td>Dilute in RPMI 5%FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LIberase TM&#160;</td>
			<td></td>
			<td></td>
			<td>300 ug/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNAse1&#160;</td>
			<td></td>
			<td style="width:155px;"></td>
			<td>50 U/ml</td>
		</tr>
	</tbody>
</table>

preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

Haut von anderen Bereich des Körpers wurde gemäß dem Protokoll, verdaut mit den angegebenen Antikörpern beschrieben und fleckig. Die Gating - Strategie ist in Abbildung 1. Wählen Sie zuerst lebende Zellen (DAPI- Zellen), dann Tor auf Singuletts (FSC-A gegen FSC-W) und auf Zellen mit Lymphozytenmorphologie (FSC-A vs. SSC-A) dargestellt. Wenn angegeben, werden CD45 + Zellen aus hämatopoetischen Ursprungs ausgewählt. DCs sind , markiert mit anti-CD11c, -MHCII und -CD45 - Antikörpern sind Makrophagen identifiziert als F4 / 80 + CD64 + CD45 + -Zellen, B - Zellen als CD19 + CD45 + MHCII + Zellen, CD4 + T - Lymphozyten , wie CD4 + CD45 + Zellen und CD8 + T - Lymphozyten als CD8 + CD45 + Zellen (Abbildung 2). Diese Methode guarantees hohe Lebensfähigkeit der verdauten Population mit mehr als 50% der gesamten lebensfähigen Zellen (DAPI-) in allen Regionen der Haut , die (3A) analysiert. Ein Hinweis auf die Anzahl der CD45 + Zellen ergeben pro cm 2 ist in 3B dargestellt. Eine Schätzung der Gesamtzahl der DCs, Makrophagen, B - Zellen und CD4 + oder CD8 + T - Zellen , die aus der Verdauung von 1 cm 2 der Haut von Mäusen ist in 3C dargestellt. <img alt="Abbildung 1" src="/files/ftp_upload/52589/52589fig1.jpg" /> Abbildung 1. Identifizierung von Zellen hämatopoetischen Ursprungs in der Hauteinzelzellsuspensionen. Einzelzellsuspensionen wurden mit DAPI und dem Anti-CD45 - Antikörper wurden gefärbt. Unter lebenden Zellen (DAPI-negativ), Unter (FSC-A gegen FSC-H) analysiert Zellen mit Leukozyten zu wählen<html> Morphologie (FSC-A vs. SSC-A). Zellen hämatopoetischen Ursprungs als CD45 + gekennzeichnet. <img alt="Figur 2" src="/files/ftp_upload/52589/52589fig2.jpg" /> Figure 2. Analyse von Immunzellen , die in der Maushaut aus verschiedenen Regionen abgeleitet. Die Zellen werden gated wie in Figur 1 angedeutet ist . DCs als CD11c + MHC II + CD45 + Zellen und später gefärbt mit CD207 identifiziert Langerhans&#39;schen Zellen und CD207 + unterscheiden dermale DCs von CD207 -; Makrophagen werden als CD64 + F4 / 80 + CD45 + Zellen identifiziert; T - Lymphozyten werden in CD8 + CD45 + Zellen und CD4 + CD45 + Zellen unterteilt; B - Zellen werden als MHCII + CD19 + CD45 + Zellen identifiziert. Prozentangaben sind auf die gesamten CD45 + Zellen bezeichnet. &quot;&gt; <img alt="Figur 3" src="/files/ftp_upload/52589/52589fig3.jpg" /> Abbildung 3. Quantitative Analyse der Immun Zellen , die in der Maushaut aus verschiedenen Regionen (A) Prozentsatz der lebensfähig. (DAPI -) Zellen stammen aus den angegebenen Bereichen der Mäusehaut. (B) Absolute Zahlen CD45 + Zellen in den verschiedenen Regionen des Mäusehaut ausgedrückt in Anzahl der Zellen pro cm 2 der Haut. (C) Schätzung Anzahl von DCs, M &amp; PHgr, B - Zellen und CD4 + oder CD8 + T - Zellen abgeleitet von 1 cm 2 der Haut von den angegebenen Regionen der Mäusehaut. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Antikörper </td><td> Die Färbung </td><td> Konzentration </td><td> Zeit </td><td> Temperatur </td></tr><tr><td colspan="5"> DCsMischen </td></tr><tr><td> CD11c </td><td> PE-Cy7 </td><td> 2 &amp; mgr; g / ml </td><td> 30 Minuten </td><td> 4 ° C </td></tr><tr><td> MHC II </td><td> PE </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD207 </td><td> APC </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td colspan="5"> makrophagen Mix </td></tr><tr><td> F4 / 80 </td><td> APC-Cy7 </td><td> 2 &amp; mgr; g / ml </td><td> 30 Minuten </td><td> 4 ° C </td></tr><tr><td> MHC II </td><td> PE </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD64 </td><td> PE-Cy7 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td colspan="5"> T und B-Zellen mischen </td></tr><tr&gt; <td> CD8a </td><td> PE </td><td> 2 &amp; mgr; g / ml </td><td> 30 Minuten </td><td> 4 ° C </td></tr><tr><td> CD4 </td><td> APC-Cy7 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD19 </td><td> APC </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr><tr><td> CD45 </td><td> PE-Cy5.5 </td><td> &quot; </td><td> &quot; </td><td> &quot; </td></tr></tbody></table> Tabelle 1 Indikationen für die Färbung der verschiedenen Populationen von Immunzellen bevölkern die Haut. Drei Mischungen angegeben sind für die Färbung von DCs, Makrophagen und Lymphozyten.

Watch this Scientific Journal Video about Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions at JoVE.com

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

Herstellung von Einzelzellsuspensionen für Cytofluorimetric Analyse von verschiedenen Mäusehaut Regionen

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

preparation-single-cell-suspensions-for-cytofluorimetric-analysis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.1K Aufrufe.  University of Milano-Bicocca. The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry. 

Serie: Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Vorbereitung der Maus Pituitary Immunogen für die Induktion der experimentellen autoimmunen Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

Forschung

Immunologie und Infektion

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Herstellung viraler DNA aus Nukleokapside

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

Granulozyten-abhängige Autoantikörper-induzierten Blasenbildung

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitative High-throughput Single-cell Zytotoxizitätstest für T-Zellen

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und<em&gt; Ex Vivo</em&gt; Zellkultur

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

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

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

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

Isolation der infiltrierenden Leukozyten aus Mäusehaut unter Verwendung enzymatischer Digest und Gradiententrennung

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

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

Herstellung von CD4<sup&gt; +</sup&gt; T-Zellen für die Analyse von GD3 und GD2 Gangliosid Membran-Expression durch Mikroskopie

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Einzellige Analyse der Immunophenotype und Produktion von Zytokinen im peripheren Blut über Masse Zytometrie

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...