乾癬患者からの皮膚生検を親切に科学的な使用のための口頭または書面によるインフォームドコンセントの後に博士アンドレアスKoerber（エッセン、ドイツの大学の皮膚科部門）によって提供されました。 XHはまた、NSFC 61263039とNSFC 11101321でサポートされています。

注：健康な個体からの皮膚生検は、科学的な使用のための口頭または書面によるインフォームドコンセントの後に選択科目整形手術を受けている個体の腹部の皮膚の残りから得ました。人間の皮膚の使用が承認されたラートボウト大学医療センター、ナイメーヘン、オランダ、エッセン、ドイツの大学の人間の研究のための医学倫理委員会によって設定された規則に従ってインチヒト皮膚からの単一細胞懸濁液の調製（フローキャビネット内の作業滅菌後、細胞培養物がある場合は必須） <ol><li>細胞培養培地を準備していない血清を含むRPMI 1640 +ペニシリン/ストレプトマイシン（ml最終濃度100単位/および100μg/ mlの、それぞれ）+ピルビン酸（0.02ミリモル）およびグルタマックス（0.02 mM）を、追加されました。 </li><li>完全培地を準備します。ステップ1.1 + 10％のヒトプール血清（HPS）で調製した培地; 4℃で保存します。私たちの前に20℃±2にメディアを持参る。 </li><li> 4ミリメートルラウンド生検パンチ装置を用いて皮膚生検を取得し、最大4時間、または4℃でONで20℃±2でRPMI1640完全培地に保管してください。実験室での到着時にできるだけ早く生検を処理します。 注：皮膚の長期保存は、細胞収量および細胞生存率に影響を与えます。 </li><li>ブルーキャップされた解離管にラベルを付け、ラベルされたチューブに5ミリリットルを完全培地を追加します。 </li><li>無菌の6ウェル培養プレート（合計3つのウェル中）を各ウェルに完全培養培地2mlを加えます。このように3回のすすぎの合計を達成し、1つのより多くの時間を、単一のウェルに生検を配置するために、無菌のツールを使用してすすぎ、第2のウェルにそれを上に移動し、このステップを繰り返します。 </li><li>滅菌シャーレによくすすぎ、皮膚生検を移し生検の上に完全培養培地100μlを追加し、慎重にステンレス鋼使い捨ての滅菌メスを使用して、皮下脂肪組織を削り取ります。 注：番目重要なステップです。 </li><li>滅菌ペトリ皿上の4小片に各皮膚生検をカット。転送サンプル（最大チューブあたり、4mmの生検の4つの）完全培養培地5mlを含有する準備解離管に。 </li><li>しっかりとキャップでチューブを閉じ、自動化された組織の解離のスリーブに上下逆さまに取り付けます。すべてのサンプル材料をロータのエリアに位置していることを確認します。 </li><li> 56秒間の適切な回転速度で生検を解離するために「プログラムm_spleenの_01」（計測器の内蔵メモリにより、または伴うプログラムカードが提供する事前定義されたプログラム）を実行することにより、解離プロセスを開始します。 </li><li>処理の後、解離から解離管を切り離し、すべての解離した材料は、チューブの底に収集されていることを確認してください。 </li><li>解離チューブに150μlのコラゲナーゼIA（80 mg / mlで）を加え、37℃で振とう水浴中でサンプルをインキュベート60分間°C。よく混ぜ、thedissociationチューブにDNアーゼI（5 MU / ml）を100μlのを追加します。 注：コラゲナーゼのより高い濃度またはより長いインキュベーション時間は、細胞生存率を変化させます。 </li><li>自動化された組織の解離のスリーブに解離管を取り付け、生検1つのより多くの時間を解離するために「 プログラムM_脾臓_01」を実行します。 </li><li> 50ミリリットルファルコンチューブの上に70μMナイロン細胞ストレーナーを配置します。細胞塊/組織片を除去するために、このセルストレーナーに解離した試料材料を適用します。 </li><li>完全培地の5ミリリットルで一回セルストレーナーを洗浄します。 20°C±2で遠心し、10分と吸引上清のための450×gで。 </li><li>洗浄工程をもう一度繰り返します。完全培養培地300μlの中に再懸濁し、細胞ペレット。単一細胞懸濁液は、さらなる分析のために準備ができています。 ex vivoでの細胞培養のためのプロトコルを続行するか、フローサイトメトリー分析。 </li><li>さらに私の場合、ntracellularサイトカイン染色は、37℃で4時間、PMA（12.5 NG /ミリリットル）、イオノマイシン（500ngの/ ml）およびブレフェルジンA（5μg/ ml）で細胞を刺激し、5％CO 2インキュベーターフローを実行する前に4時間フローサイトメトリー分析。 </li></ol>皮膚常駐T細胞の2ポリクローナル活性化（ex vivoで細胞培養） <ol><li>アリコート（ステップ1.15で調製した）100μlの単一細胞懸濁液を、丸底96ウェルプレートに。 注：4mMの皮膚生検のそれぞれについて、取得された単一細胞懸濁液を96ウェルプレートのうちの少なくとも2つのウェルに分割することができます。 </li><li>抗CD3 /抗CD28 mAbで被覆したマイクロビーズ（ウェル当たり25,000ビーズ）、組換えヒトサイトカインのrIL-2（最終濃度25 U / ml）およびのrIL-1β（最終濃度50 ngの/ mlで）を加えます。 </li><li>ウェル標識プレートの蓋で培養プレートをカバーし、5％CO 2、100％湿度でインキュベートし、37 Cのインキュベーター°。培地の色が黄色になるとメディアを変更します。 <br/&gt;注：明細胞コロニーは、細胞培養の8日目から観察することができます。 </li></ol>プライマリ/培養皮膚常駐T細胞の3フローサイトメトリー分析<ol><li> PBS + 0.2％BSA：FACS緩衝液を準備します。 </li><li> V底96ウェルプレートに、目的とする細胞を転送します。 20°C±2、2分間、450×gで遠心し、その上清を吸引。 </li><li> 100μlのPBSにペレットを再懸濁。 20°C±2、2分間、450×gで遠心し、その上清を吸引。 </li><li> 4℃で30分間：（PBSを使用して、千希釈1）固定可能な生存可能性色素を結合させ準備eFluorescence780100μlの染色細胞。 FACS緩衝液100μl、20℃±2で遠心分離機、2分、および吸引上清450 XGを追加します。 </li><li>例えば、必要な細胞表面マーカーのmAbを選択：CD45-BV421（HI30、1時20分）、CD3-ECD（UCHT1、午前1時50分）、CD4-PC5.5（1388.2、1：200）、CD8-APC-AlexFluo700 （B9.11、1：400）、CD14-FITC（TUK4、1時50分）、CD19-APC-AlexFluo750（13から1191:50）、CD56-PE（MEM-188、1：50）、CD25-PeCy7（BC96、1時50分）、のCD11c-PeCy7（BU15、1:10）、およびCD1c-APC（AD5-8E7、 1:10）、テストした各mAbの希釈率に応じて、FACS緩衝液を使用したmAb-混合物を準備します。非染色サンプルと一緒に、抗体のアイソタイプコントロールを使用して、ゲートの設定を定義します。 </li><li>各ウェルに準備したmAb-混合物の25μlのを追加します。光から保護し、20℃±2で20分間インキュベートします。 </li><li> FACS緩衝液100μl、20℃±2で遠心分離機、2分、および吸引上清450 XGを追加します。 </li><li>唯一の細胞表面染色を必要とするサンプルについては、ステップ3.16に進みます。 </li><li>細胞内のFoxp3染色の場合： <ol><li>希釈剤の3部で濃縮物の一部を混合することによって固定し、透過処理バッファーを準備します。 </li><li> 10倍の透過化バッファの1部+滅菌H 2 Oの9部：1×透過処理バッファーを準備します</li></ol></li><li>固定とpermeabiliの100μl中に再懸濁ペレット化バッファは、よく混ぜ、および4でインキュベート 30分間°C。 </li><li>各ウェルに450 2分間、室温でXG、および吸引上清で遠心分離機を100μlの透過化バッファを追加します。 </li><li>透過化バッファ1より多くの時間で細胞を洗浄。 </li><li>例えば、必要な細胞内のmAbを、選択し、Foxp3を-eFluo450（PCH1​​01、1：50）、IL-17A-AlexFluo488（eBio64DEC1、1：50）およびIFNγ-PeCy7（4S.B3、1：400）。テストした各mAbの希釈倍率に応じて、2％正常ラット血清を含む透過化緩衝液を用いてモノクローナル抗体の混合物を準備します。非染色サンプルと一緒に、抗体のアイソタイプコントロールを使用して、ゲートの設定を定義します。 </li><li>各ウェルに準備したmAb-混合物の20μlのを追加します。 4インキュベート 30分間°Cで、光から保護します。 </li><li> 30分間のインキュベーション後、各ウェルに透過化緩衝液100μlを加えます。 20°C±2、2分間、450×gで遠心し、その上清を吸引。 </li><li>透過性のブフェで細胞を洗浄R 1のより多くの時間。再懸濁し、FACS緩衝液の110μlのでペレット、およびmicroFACSチューブに細胞懸濁液を転送します。 注：サンプルは、10色のフローサイトメトリーを用いた測定の準備ができています。 </li><li>必要とされる正確な細胞数の場合には、直ちにフローサイトメトリーを用いて測定を行う前に、各試料中に蛍光粒子の十分に混合された均一な懸濁液10μlを追加します。 </li></ol>

<ol>
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The authors have nothing to disclose.

ここでは、効率的にヒトの皮膚生検からの皮膚常在T細胞を単離するためのプロトコルを提示します。このプロトコルの利点は、実行可能なリンパ球の比較的高い数の分離であり、関連する表面マーカーを発現します。同定された細胞のサブセットは以下の通りであった：たCD11c + DCは、CD4 +およびCD8 + T細胞とのFoxp3 + CD25 +細胞。重要なのは、孤立した?...

皮膚は、身体と環境との間の主要なインタフェースとして、外部からの物理的、化学的、およびそのような創傷、紫外線や微生物などの生物学的傷害に対する防御の最初の行を提供します。皮膚は、主に2つのコンパートメント、表皮と真皮を含み、ランゲルハンス細胞、マクロファージ、樹状細胞（DC）、および約200億メモリーT細胞を含む免疫細胞の様々な含まれ、全体の血液量1、ほぼ2倍の数の本、2。データの成長体は、皮膚が組織の恒常性の間、および様々な病理学的状態の両方で、本質的な免疫学的機能を持っているという概念をサポートしています。正常な皮膚内に存在する免疫細胞を、免疫学的監視3を実施するために考えられ、例えば、乾癬4のような炎症性疾患の発症において役割を果たすことが示されています。乾癬病変皮膚では、CD4 +およびCD8 +の両方のT細胞がOBSた浸潤しましたervedそれはCD4とCD8の比率は、疾患状態5に応じて変化することが示されました。しかし、細胞のこの集団は、既存の技術は、ほんのわずかの細胞の単離を可能にするため、研究するのが困難です。 ヒトの皮膚からのT細胞の単離のために現在広く用いられている技術は、酵素処理と機械的に皮膚解離を兼ね備えています。ヒトの皮膚生検は、広範囲に刻み、トリプシン、コラゲナーゼおよび/ ​​またはEDTA 6-8のような酵素でインキュベートします。皮膚は、引張力および機械的分解に非常に抵抗性であるバリア組織であることを考慮すると、T細胞の単離の確立された方法は、これらの細胞集団の困難およびex vivo細胞培養を行い、非常に少数の細胞および生細胞のより低い番号を生成します挑戦。 ここでは、mechanを組み合わせることにより、健康で関与両方乾癬ヒトの皮膚からリンパ球を単離するための改良法を報告します酵素消化は、コラゲナーゼを使用して一緒に、自動化された組織の解離の代わりに広くミンチの確立された方法を用いて、皮膚のiCalの解離。 DCおよびT細胞を含む種々の生存免疫細胞サブセットを、単一細胞懸濁液の調製後に観察されました。重要な表面マーカーCD3、CD4とCD8の発現がよく保存されていました。このようにして調製した細胞は、ex vivoでの細胞培養またはフローサイトメトリー分析に使用するための準備ができています。このプロトコルは、正常乾癬患者の病変皮膚に由来する単一の皮膚生検（4 mm）での分析のために使用されています。その結果、皮膚常駐患者のT細胞は、健康なボランティア9と比較して、IL-17及びIFNγのような多くの炎症性サイトカインを産生することを示しました。

<table><tbody><tr><td>Disposable Biopsy Punch</td><td>Kai Europe</td><td>BP-40F</td><td>4 mm</td></tr><tr><td>Disposable sterile scalpels</td><td>Dalhausen</td><td>1100000510</td><td></td></tr><tr><td>gentleMACS C tube</td><td>Miltenyi Biotech</td><td>130-093-237</td><td>Blue-capped, used as the dissociation tube.</td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotech</td><td>130-093-235</td><td>Automated tissue dissociator. Using the &quot;program spleen_01&quot; for dissociation of the skin biopsy.</td></tr><tr><td>Cell strainer&amp;nbsp;</td><td>BD</td><td>352350</td><td>70 &amp;micro;m nylon</td></tr><tr><td>96-well U-bottom plate</td><td>Greiner Bio-One</td><td>650180</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>22409-015</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Human Pooled Serum (HPS)</td><td>in house prepared</td><td></td><td></td></tr><tr><td>Collagenase I</td><td>Sigma-Aldrich</td><td>C2674</td><td>Type 1-A, suitable for cell culture</td></tr><tr><td>DNase I</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>PBS</td><td>B Braun</td><td>3623140</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A4503-500G</td><td></td></tr><tr><td>Fixable Viability Dye (FVD) APC-eFluo780</td><td>eBioscience</td><td>65-0865-18</td><td>Stain dead cells prior to cell fixation; dilute with PBS at 1:1,000</td></tr><tr><td>Fixation/Permeabilization Concentrate</td><td>eBioscience</td><td>00-5123-43</td><td></td></tr><tr><td>Fixation/Permeabilization Diluent</td><td>eBioscience</td><td>00-5223-56</td><td></td></tr><tr><td>Permeabilization Buffer (10x)</td><td>eBioscience</td><td>00-8333-56</td><td></td></tr><tr><td>BV421 Mouse anti-human CD45</td><td>BD</td><td>563879</td><td>Clone: HI30; dilution factor 1:50</td></tr><tr><td>FITC Mouse anti-human CD14</td><td>Dako</td><td>T0844</td><td>Clone: TUK4; dilution factor 1:50</td></tr><tr><td>PE Mouse anti-human CD56</td><td>Dako</td><td>R7127</td><td>Clone: MOC-1; dilution factor 1:50</td></tr><tr><td>ECD Mouse anti-human CD3</td><td>Beckman - Coulter</td><td>A07748</td><td>Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td></tr><tr><td>PC5.5 Mouse anti-human CD4</td><td>Beckman - Coulter</td><td>B16491</td><td>Clone: 13B8.2; dilution factor 1:200</td></tr><tr><td>PeCy7 Mouse anti-human CD11c</td><td>Beckman - Coulter</td><td>A80249</td><td>Clone: BU15; dilution factor 1:50</td></tr><tr><td>APC Mouse anti-human CD1c</td><td>Miltenyi Biotech</td><td>130-090-903</td><td>Clone: AD5-8E7; dilution factor 1:10</td></tr><tr><td>APC-AlexFluo700 Mouse anti-human CD8</td><td>Beckman - Coulter</td><td>A66332</td><td>Clone: B9.11; dilution factor 1:400</td></tr><tr><td>APC-AlexFluo750 Mouse anti-human CD19</td><td>Beckman - Coulter</td><td>A94681</td><td>Clone: J3-119; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human CD25</td><td>eBioscience</td><td>AD5-8E7</td><td>Clone: BC96; dilution factor 1:50</td></tr><tr><td>&amp;nbsp;PE Rat anti-human CLA</td><td>Miltenyi Biotech</td><td>130-091-635</td><td>clone: HECA-452; dilution factor 1:</td></tr><tr><td>eFluo450 Rat anti-human Foxp3</td><td>eBIoscience</td><td>48-4776-42</td><td>Clone: PCH101; intracellular staining; dilution factor 1:50</td></tr><tr><td>AlexFluo488 Mouse anti-human IL-17A</td><td>eBioscience</td><td>53-7179-42</td><td>Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td></tr><tr><td>PeCy7 Mouse anti-human IFNg</td><td>eBioscience</td><td>25-7319-41</td><td>Clone: 4S.B3; intracellular staining; dilution factor 1:400</td></tr><tr><td>Flow-Count Fluorospheres</td><td>Beckman - Coulter</td><td>7547053</td><td>Counting beads, for flow cytometry</td></tr></tbody></table>

lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture

人間の皮膚は重要なバリア機能を有しており、病原体からの組織の恒常性と保護に貢献様々な免疫細胞が含まれています。肌がアクセスすることは比較的容易であるように、それは末梢免疫制御メカニズムを研究するための理想的なプラットフォームを提供します。免疫常駐健康な皮膚行動免疫監視中の細胞だけでなく、乾癬のような炎症性皮膚疾患の発達において重要な役割を果たしています。新興洞察力にもかかわらず、様々な炎症性皮膚疾患の根底にある生物学の我々の理解はまだ限られています。生検皮膚試料から単離された良質な（単一）細胞集団が必要です。これまでに、単離手順は、深刻な生存細胞の十分な数を得ることの欠如によって妨げられてきました。単離およびその後の分析にも得ることが、現在の解離手順によって引き起こされる機械的及び化学的ストレスに起因する免疫細胞系統マーカーの損失の影響を受けています単一細胞懸濁液。ここでは、自動化された組織の解離及びコラゲナーゼ処理を用いた機械的皮膚解離を組み合わせることにより、健康と関係する両方の乾癬のヒト皮膚からT細胞を単離するために修正された方法を記載します。この方法は、単一細胞懸濁液を調製する際に、このようなCD4、CD8、Foxp3をおよびCD11cなど、ほとんどの免疫系統マーカーの発現を保持します。成功したCD4 + T細胞の単離およびその後の表現型および機能的分析の例が示されています。

単4ミリメートルの皮膚生検を使用している場合、ここで提示されたプロトコルは、ヒトの皮膚から（±SEM、乾癬患者の病変皮膚を意味する）760±178,000まで（±SEM、健康なボランティアの皮膚を意味する）615±2200の間に実行可能なリンパ球を得られます。 CD45 +細胞の異なる種類はCD4 + T細胞（〜45％）、CD8 + T細胞（約30％）を含む、健康な個体の皮?...

Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

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 ...

lymphocyte-isolation-from-human-skin-for-phenotypic-analysis-ex-vivo

Research

JoVE Journal

Immunology and Infection

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

14.5K ビュー.  Radboud University Medical Centre. 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.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

移行のヒトTリンパ球の単離、培養および解析インビトロ</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

免疫・感染学

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

蛍光活性化細胞選別によるヒトの皮膚からの乳頭状および網状線維芽細胞の単離

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

遺伝学

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

マウス生殖管粘膜からのリンパ球の単離

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

単離とマウスリンパ球の活性化

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

医学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

生物学

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

ヒト包皮からの初代シュワン細胞、ケラチノサイト、および線維芽細胞の単離、培養、および特性評価

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

神経科学

Human Skin Keratinocyte Isolation: A Method to Culture Primary Keratinocytes from Skin Samples

Johansen, C. et al. <a href="https://www.jove.com/v/56863"> Generation and Culturing of Primary Human Keratinocytes from Adult Skin</a>. J. Vis. Exp. (2017)
This video describes the isolation of keratinocytes&#8212;the most prominent cells of human skin&#39;s epidermal layer, by employing a combination of enzymatic digestion and mechanical disruption methods. This method helps in the preferential growth of keratinocytes over other skin cell types using a selective growth medium.

ヒト皮膚ケラチノサイト単離:皮膚サンプルから初代ケラチノサイトを培養する方法

Johansen, C. et al.  Generation and Culturing of Primary Human Keratinocytes from Adult Skin. J. Vis. Exp. (2017)
This video describes the isolation of ...

表現型分析のためのヒト皮膚からリンパ球単離と

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移行のヒトTリンパ球の単離、培養および解析インビトロ