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.

研究の承認：実験プロトコルはDecretoに応じた保健のイタリア省（ローマ、イタリア）によって承認された27 gennaio 1992 legislativo、n個。 116「AttuazioneデッラDirettivaのn。609分の86 / CEEでマテリアディprotezioneデッリanimali utilizzati Aフィニsperimentali O広告altriフィニscientifici。」 1.皮膚サンプルを入手<ol><li>頸椎脱臼によりマウスを安楽死させます。 </li><li>耳： <ol><li>マウスの耳の毛のない部分を切り取ります。 </li><li>鉗子でそれらを引き離すことにより、独立した背と腹側。静かにピンセットで耳の内側の部分をこすることによって、残りの軟骨を取り除きます。 </li></ol></li><li>トランク皮膚： <ol><li>電気かみそりで、繊細な脱毛ローションで両方の動物の皮膚全体を剃ります。 </li><li> （2 cm 2で全動物の皮膚まで）所望の皮膚サンプルを遮断。 <ol><li>全体の背側を切断した場合とVENクトルのマウスの皮膚は、バックした後、マウス本体の周りの毛を剃った領域の境界線に沿って切断し、マウスの真ん中の縦のカットを行います。 </li><li>静かに手術用ハサミやメスの閉じラウンドエッジの先端で皮膚のサンプルを分離しながら、鉗子でまだ皮膚サンプルを保ち、腹膜や背中の筋肉から皮膚を分離します。 </li></ol></li><li>氷冷PBSの10ミリリットルを含む100ミリメートル細胞培養プレートを準備します。 </li><li>ステップ1.3.3で製造したプレートにPBSで皮膚サンプルを浸漬し、鉗子で皮膚試料をこすることによって皮下脂肪を除去します。 </li></ol></li><li>尾： <ol><li>マウスの尾を切り落としました。 </li><li>尾の基部から始まり、尾の先端に到達し、手術用はさみまたは外科メスで尾の垂直カットを行います。 </li><li>尾から皮膚を分離するために鉗子を使用してください。尾の基部にカットのエッジをつかみ、そっと押さえながら先端に向かって引っ張ります鉗子で尾の体。 </li><li>優しく皮膚試料をこすることによって、任意の余分な脂肪を取り除きます。 </li></ol></li><li>追いはぎ<ol><li>手術用ハサミでマウス全体の足を切り取ります。任意の有毛皮膚を切断回避。 </li><li>数字の先頭に縦方向に足首から足の皮膚をカット。 </li><li>鉗子で皮膚をつかんながら鉗子でまたは手で足首の骨を持ち、手袋を脱いでいるかのように数字に向かって皮膚を引っ張ります。 </li></ol></li></ol> 2.消化<ol><li>酵素ミックス（仕様のための材料リストを参照してください）​​300μgの/ mlの、DNAse1 50 U / mlの次のレシピ（材料リストのように調製）ストック溶液から消化カクテルを準備します。 RPMI 5％FBS中で希釈します。 </li><li> （：消化カクテルの2ミリリットル皮膚のあらゆる2 cm 2で、最大3 mlの全トランク肌用）消化カクテルの2ミリリットルと35ミリメートルペトリ皿に小片にハサミで皮膚サンプルをみじん切りに。 </li><li>90分間37℃でインキュベートします。ゆっくりインキュベーション中のペトリ皿に1-2回振ります。 </li><li> RPMI1640 5％FBSの1ミリリットルを含む66ミリメートルペトリ皿に70μmのセルストレーナーに消化された皮膚試料を注ぎます。 </li><li> RPMI 1640の5ミリリットルの5％FBSでストレーナーを洗ってください。 </li><li>シリンジプランジャと細胞ストレーナーを通して試料を絞ります。 </li><li>フラッシュプランジャー、RPMI1640 5％FBSの20ミリリットルとペトリ皿とストレーナ、50ミリリットルチューブに移します。 </li></ol> 3.抗体とラベリング<ol><li> PBS、1％FBS（またはBSA）の5ミリリットルで二回得られた懸濁液を洗​​ってください。 </li><li>所望の抗体で得られた細胞懸濁液にラベルを付けます。 <ol><li> 100μlのサンプルを再懸濁/ PBS、1％FBS中で希釈したaCD16 / CD32（クローン2.4G2）を2μg/ mlを含むミックスのサンプル。 </li><li> Fc受容体を介して、所望の抗体の非特異的結合をブロックするために4℃で15分間インキュベートします。 </li><li> 表に示した抗体を追加します。1。 <ol><li> 4μg/ mlの濃度でPBS、1％FBSを100μl/サンプル中の所望の抗体を希釈ミックスを調製します。 </li><li>チューブに、2μgの/ mlの最終濃度で抗体混合物200μlの容量が存在することになるように、各サンプルにステップ3.2.3.1で準備ミックス100μlのを追加します。 </li></ol></li><li> 4℃で30分間インキュベートし、光から覆います。 </li></ol></li><li> PBS、1％FBSの1ミリリットル、400×gで5分間の遠心分離で洗浄し、PBS、1％FBSの300μlの上清および再懸濁を捨てます。 </li><li> 2μg/ mlの最終濃度でDAPIを加え、10分間、光から保護し、4℃でインキュベートします。 </li><li> FACSによって得られた試​​料を分析します。 <ol><li>白血球を単離するために、以下のゲーティング戦略を使用します。 <ol><li>まずDAPI-集団に対してゲートすることによって、任意の死細胞を排除します。 </li><li> 図1に示すように、FSCのWプロット対FSC-Aにシングレットを選択</strong&gt;。 </li><li>ゲート関心の白血球の典型的な大きさと細かさに基づいてセル。 </li><li> 図1に示すようにFSC-CD45プロットでCD45 +細胞を選択します。 </li></ol></li><li>次のように、表面マーカーの発現によって、白血球の異なる集団を区別する。 <ol><li> CD11c + MHC II +細胞としてDCを識別します。これらは、後でCD207-皮膚樹状細胞からのランゲルハンス細胞及びCD207 +皮膚樹状細胞を区別するためにCD207の発現について分析することができます。 </li><li> CD64 + F4 / 80 +細胞としてマクロファージを識別します。 </li><li> CD19 + MHCII +細胞などのB細胞を識別します。 </li><li> CD8 +またはCD4 +細胞のようなT細胞を識別します。 </li></ol></li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我々は、異なるマウス皮膚領域からの単一細胞懸濁液を調製するための方法を記載しています。我々が採用した消化の方法は、高い収率を与えるだけでなく、その後のFACS分析のために基本的な表面マーカーの発現を維持するだけでなく。 コラゲナーゼIおよびIIおよびサーモリシンのカクテルの使用は、この方法は、再現性の高い作り酵素活性のバッチの違いに最小バッチを保証します。他の公表された方法は、異なる酵素カクテルに依存しているが、我々の手で、彼らはより低い収率を与えると、もっと重要なのは、あまり再現性があります。私たちの方法は、しかし、表皮の移植細胞と真皮を移植する細胞を区別しません。多くの場合、それは、これら2つの異なる区画を区別することが有用である可能性があるため、我々はこのような方法を使用して、これらの以前の作品を読者に指示します。15 我々の経験の皮膚試料中の耳と尾からの皮下グリース層は、徹底的に酵素カクテルに真皮にアクセスできるようにするために掻き取られなければならないトランク皮膚よりも消化しやすくなります。脱加脂工程、並びに切断は細胞の生存を維持するために、迅速かつ静かに行うべきであるけれども。長期化または脱脂工程は、皮膚内に常駐免疫細胞に損傷を与えるので、はるかに低い収量をもたらし、回収された細胞の生存率を低下させることができる、あまりにも徹底。 消化は、βメルカプトエタノールなどの還元剤を欠く培地中で行うべきで、表面マーカーの完全性を維持することもを留意すべきです。マーカー表面への不要な損傷を回避するために、インキュベーション時間は、できるだけ短くとして保たれるべきであり、いずれにしても、以上90分間延長しません。 細胞内に消化された皮膚サンプルを注ぐときに、特に細心の注意を、また、取られるべきですストレーナ。徹底的にシリンジプランジャで消化されたサンプルを壊し前に遊離した細胞を洗い流すために、消化皮膚を洗浄することが重要です。このステップでは皮膚サンプルは、任意のより多くの物理的なストレスを受けない遊離した免疫細胞から洗浄されます。スマッシングは、任意の細胞を死滅させないために穏やかに行われるべきであり、セルストレーナーとシリンジプランジャの両方が完全に続く洗浄工程で洗浄されるべきです。 皮膚の消化はまた、37℃の水浴中で、50mLのファルコンで行うことができます。この場合、スキンは、予め細断することができ、その後、ファルコンチューブに入れました。皮膚試料は常にインキュベーション中に消化緩衝液中に沈めなければならず、その耳片がファルコンチューブの側面に固執することに注意してください。ファルコンチューブを使用する場合は、したがって、消化培地に沈めみじん切り皮膚料金の作品を確認してください。 この方法は、ミリアンペアに役立ちます特に非炎症を起こした皮膚を研究nyの異なる実験の設定、。炎症、血管拡張および組織の間に血液から組織および免疫細胞の構造を緩める腫れ大規模な肌に補充されます。細胞回収の効率は、従って、非常に高いです。恒常性条件下で、真皮のタイトコラーゲンマトリックスを消化するための効果的な方法は、実際には、皮膚を移入複雑なネットワークを構成する免疫細胞を回収するために必要とされます。 また、読者は、恒常性および炎症性の両方の条件に皮膚を移植異なる樹状細胞およびマクロファージの起源と表現型をカバーする包括的なレビューに興味があるかもしれません。16 別の実験設定では、マウスの皮膚の異なる部分を処理することができます。例えば、皮膚移植モデルで、尾の部分が受けた動物の胴体に移植することができます。 studyinとき耳はしばしばDTHアッセイでT細胞記憶を評価するために使用されるG浮腫形成または免疫化手順は、刺激は、多くの場合、マウスの足蹠に注射します。私たちの方法で、私たちは、効率的な選択の実験設定のための私達の方法が有用となるの異なる場所から皮膚常駐免疫細胞を抽出することもできます。

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>
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			<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.

体のさまざまな部位からの皮膚は、標識した抗体を用い説明し、染色されたプロトコルに従って消化しました。ゲーティング戦略は、まずシングレット（FSC-W対FSC-A）にし、リンパ球の形態（SSC-A対FSC-A）を持つ細胞でライブ後、細胞（DAPI-細胞）、ゲートを選択し、図1に示されています。示された場合に、造血起源のCD45 +細胞が選択されます。 DCは、マクロファージは、F4 / 80 + CD64 + CD45 + CD4 + CD45 +のようなCD19 + CD45 + MHCII +細胞、CD4 + Tリンパ球のような細胞、B細胞として同定される、抗CD11cの、-MHCII、および-CD45抗体で標識されていますCD8 + CD45 +細胞（ 図2）のような細胞、およびCD8 + Tリンパ球。 このメソッド区分析された皮膚の全ての領域における総生存細胞（DAPI-）の50％以上で消化人口（ 図3A）のarantees高い生存性。 CD45 +細胞の数の表示は、 図3Bに示されている1cm 2当たりもたらします。 マウス皮膚の1cm 2の消化から得られたDC、マクロファージ、B細胞およびCD4 +またはCD8 + T細胞の総数の推定値は、図3Cに示されています。 <img alt="図1" src="/files/ftp_upload/52589/52589fig1.jpg" /> 皮膚の単細胞懸濁液中の造血起源の細胞の図1の同定。単一細胞懸濁液を、DAPIおよび抗CD45抗体で染色されています。生細胞（DAPI陰性）の中で、シングレット（FSC-H対FSC-A）は白血球で細胞を選択するために分析されます<html>形態（SSC-A対FSC-A）。造血起源の細胞は、CD45 +として同定されます。 <img alt="図2" src="/files/ftp_upload/52589/52589fig2.jpg" /> 異なる領域に由来するマウスの皮膚に存在する免疫細胞の図2.分析。 図1に示すように、細胞がゲートされる。DCはのCD11c + MHC II + CD45 +細胞として同定され、それ以降のランゲルハンス細胞を区別するためにCD207で染色し、CD207 +されていますCD207から真皮のDC - 。マクロファージは、CD64 + F4 / 80 + CD45 +細胞として同定されます。 Tリンパ球は、CD8 + CD45 +細胞およびCD4 + CD45 +細胞に分割されます。 B細胞は、MHCII + CD19 + CD45 +細胞として同定されます。パーセンテージは、総CD45 +細胞と呼ばれています。 &quot;&gt; <img alt="図3" src="/files/ftp_upload/52589/52589fig3.jpg" /> 異なる地域からのマウスの皮膚に存在する免疫細胞の図3.定量分析 、生存の（A） の割合。（DAPI - ）マウス皮膚の示す領域に由来する細胞。マウス皮膚の異なる領域における（B）の絶対数のCD45 +細胞は、皮膚の1cm 2当たりのセルの数で表されます。 DCの（C）を推定数、MΦ、B細胞とマウス皮膚を示した領域から皮膚の1cm 2の由来するCD4 +またはCD8 + T細胞。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 抗体 </td><td> 染色 </td><td> 濃度 </td><td> 時間 </td><td> 温度 </td></tr><tr><td colspan="5"> DCはミックス</td></tr><tr><td> CD11c </td><td> PE-Cy7の</td><td> 2 / mlの</td><td> 30分</td><td> 4°C </td></tr><tr><td> MHC II </td><td> PE </td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD45 </td><td> PE-Cy5.5を</td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD207 </td><td> APC </td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td colspan="5">マクロファージミックス</td></tr><tr><td> F4 / 80 </td><td> APC-Cy7を</td><td> 2 / mlの</td><td> 30分</td><td> 4°C </td></tr><tr><td> MHC II </td><td> PE </td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD45 </td><td> PE-Cy5.5を</td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD64 </td><td> PE-Cy7の</td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td colspan="5"> TおよびB細胞のミックス</td></tr><tr&gt; <td> CD8a </td><td> PE </td><td> 2 / mlの</td><td> 30分</td><td> 4°C </td></tr><tr><td> CD4 </td><td> APC-Cy7を</td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD19 </td><td> APC </td><td> 「 </td><td> 「 </td><td> 「 </td></tr><tr><td> CD45 </td><td> PE-Cy5.5を</td><td> 「 </td><td> 「 </td><td> 「 </td></tr></tbody></table> 皮膚を移植免疫細胞の異なる集団の染色表 1. 表示はスリーミックスはDCを、マクロファージおよびリンパ球の染色のために示されています。

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.

別のマウス皮膚の領域から細胞蛍光分析のための単一細胞懸濁液の調製

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 ビュー.  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. 

ビデオ: 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.

実験的自己免疫性下垂体炎の誘導のためのマウス下垂体免疫原の調製

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

免疫・感染学

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.

ヌクレオカプシドからウイルスDNAの調製

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.

の単一細胞解析枯草菌</em蛍光顕微鏡とフローサイトメトリーを用いて>バイオフィルム

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.

顆粒球に依存する自己抗体誘発皮膚水疱形成

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.

T細胞の定量的なハイスループットシングルセル細胞毒性アッセイ

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.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

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.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

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.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

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.

CD4の調製<sup&gt; +</sup&gt;顕微鏡によるGD3およびGD2ガングリオシドの膜発現の分析のためのT細胞

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.

イムノフェノ タイプと大量のフローサイトメトリーによる末梢血中のサイトカイン産生の単一細胞解析

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