Sedef hastalarının deri biyopsileri nazik Dr. Andreas Koerber bilimsel kullanım için yazılı veya sözlü aydınlatılmış onam sonra (Essen, Almanya Üniversitesi Dermatoloji bölümü) tarafından verilmiştir. XH da NSFC 61263039 ve NSFC 11101321 tarafından desteklenmektedir.

NOT: sağlıklı bireylerden deri biyopsileri, bilimsel kullanım için yazılı veya sözlü aydınlatılmış onam sonra elektif plastik cerrahisi geçiren bireylerin karın cilt leftover elde edilmiştir. insan derisinin kullanımı onaylanmış ve oldu Radboud üniversite tıp merkezi, Nijmegen, Hollanda ve Almanya&#39;nın Essen Üniversitesi insan araştırma Tıbbi Etik Komiteleri tarafından belirlenen yönetmeliklere uygun olarak. İnsan Cilt gelen Tek Hücre süspansiyonları 1. Hazırlık (a Flow-kabinede Çalışma Steril sonraki Hücre Kültürü ise Gerekli) <ol><li> Hücre kültürü ortamı hazırlayın: Resim serumu RPMI 1640 + Penisilin / streptomisin (ml nihai konsantrasyonları, 100 ünite / 100 ug / ml) ile + piruvat (0.02 mM) ve Glutamax (0.02 mM) ilave edildi. </li><li> Tüm kültür ortamı hazırlayın: kültür ortamı, adım 1.1 +% 10 insan toplanan serumdan (HPS) hazırlanmıştır; 4 ° C&#39;de saklayın. Bizden önce 20 ° C ± 2 orta getiring. </li><li> Bir 4mm yuvarlak biyopsi yumruk aleti kullanılarak deri biyopsisi elde edilir ve en fazla 4 saat veya 4 ° C AÇIK 20 ° C ± 2 de 1640 tam kültür ortamında saklayın. Laboratuvarda girişte en kısa sürede biyopsi işleyin. NOT: Derinin uzun depolama hücresi verim ve hücre canlılığı etkileyecektir. </li><li> mavi kapaklı ayrışma tüp Etiket ve etiketli tüpe 5 ml tam kültür ortamı ekleyin. </li><li> steril 6 oyuklu kültür plakasının (toplam 3 delikleri şeklinde), her oyuğa tam kültür ortamı içinde 2 ml ilave edilir. böylece üç durulama toplam elde, tek bir kuyunun içine biyopsi yerleştirmek durulayın, ikinci kuyunun onu taşımak ve bu adımı bir kez daha tekrar etmek steril araçlarını kullanın. </li><li> Steril Petri kabı iyi durulanmalıdır deri biyopsisi transfer biyopsi üstünde tam kültür ortamı 100 ul ekleyin ve dikkatlice bir paslanmaz çelik tek kullanımlık steril neşter kullanılarak deri altı yağ dokusu kazıyın. NOT: Tholan kritik bir adımdır. </li><li> Steril bir Petri kabı 4 küçük parçalar halinde her cilt biyopsisi kesin. Aktarım örnekleri (kadar tüp başına 4 mm biyopsi dört) tam kültür ortamı içinde 5 ml ihtiva eden hazırlanan ayrıştırma tüpüne. </li><li> Sıkıca kapaklı tüpler kapatın ve otomatik doku ayrıştırıcı kovanına ters takın. Tüm örnek malzeme rotor bölgede yer olduğundan emin olun. </li><li> &quot;Program m_spleen _01&quot; 56 sn için uygun döner hızında biyopsi ayırmak (birlikte bir program kartına cihazın dahili hafızasında veya tarafından sağlanan önceden tanımlanmış bir program) çalıştırarak ayrılma işlemini başlatın. </li><li> işlendikten sonra, dissociator gelen ayrılma tüpü ayırmak ve tüm ayrışmış madde tüpün dibinde toplanan olduğundan emin olun. </li><li> 37 ° C&#39;de çalkalanan bir su banyosu içerisinde ayrıştırma tüpüne kolajenaz IA (80 mg / ml) ul 150 ilave edin ve örnek inkübe60 dakika ° C. thedissociation tüpüne DNaz I, 100 ul (5 MU / ml) ekleyin, iyice karıştırın. Not: kolajenaz ya da hücre canlılığında değiştirecek uzun kuluçka süresi yüksek konsantrasyonlarda eklenmiştir. </li><li> Otomatik doku ayrıştırıcı kovana ayrışma tüp takın ve Biyopsiyi bir kez daha ayırmak &quot;programı m_ dalak _01&quot; çalıştırın. </li><li> bir 50 ml Falcon tüpü üzerine 70 um naylon hücre süzgecinden yerleştirin. hücre kümeleri / doku enkaz kaldırmak için bu hücre süzgeç ayrışmış örnek malzemeleri uygulamak. </li><li> Tüm kültür ortamı, 5 ml ile bir kez hücre süzgecinden yıkayın. 20 ° &#39;de santrifüje C ± 2, 10 dakika ve aspirat yüzer 450 x g. </li><li> Yıkama adımı bir kez daha tekrarlayın. tam kültür ortamı içinde 300 ul tekrar süspansiyon hücre peletleri. Tek hücre süspansiyonları daha fazla analiz için hazırdır; ex vivo hücre kültürü için protokollerle devam etme veya akış sitometri analizine. </li><li> Daha fazla i halindentracellular sitokin boyama, 37 ° C de 4 saat PMA (12.5 ng / ml), İyonomisin (500 ng / ml) ve Brefeldine A (5 ug / ml) ve hücreleri uyarır,% 5 CO2 kuluçka akış uygulamadan önce 4 saat sitometri analizi. </li></ol> Cilt Resident T hücrelerinin 2. Poliklonal Aktivasyon (Ex Vivo Hücre Kültürü) <ol><li> Yuvarlak dipli 96 gözlü plaka içine kısım 100 ul tek hücre süspansiyonları, (adım 1.15 hazırlandı). Not: 4 mm deri biyopsisi her biri için, elde edilen tek hücre süspansiyonları, 96 oyuklu bir plakanın en az iki kuyu ayrılabilir. </li><li> anti-CD3 / anti-CD28 mAb ile kaplanmış mikrotaneler (oyuk başına 25,000 boncuk), rekombinant insan sitokin rlL-2 (nihai konsantrasyon 25 U / ml) ve rlL-1β (nihai konsantrasyon 50 ng / ml) eklenir. </li><li> % 5 CO2,% 100 nem, 37 inkübe iyi olarak işaretlenmiş levha kapak kültürü plakası Kapak C inkübatör °. orta renk sarıya döndüğünde orta değiştirin. <br/&gt; Not: Açık bir hücre kolonisi hücre kültürünün 8 gün arasında gözlemlenebilir. </li></ol> 3. İlköğretim Sitometrisi Analizi / kültürlü Cilt Resident T hücrelerinin <ol><li> PBS +% 0.2 BSA: FACS tampon hazırlayın. </li><li> V-tabanlı 96 oyuklu bir plakaya ilgi hücreleri aktarın. 20 ° C ± 2, 2 dakika boyunca 450 x g&#39;de santrifüj ve aspire yüzer. </li><li> 100 ul PBS içinde pelletini. 20 ° C ± 2, 2 dakika boyunca 450 x g&#39;de santrifüj ve aspire yüzer. </li><li> (: PBS kullanarak 1000 sulandırma 1), 4 ° C&#39;de 30 dakika boyunca sabitlenebilir canlılığı boya konjüge hazırlanmış eFluorescence780 100 ul Leke hücreleri. 20 ° C ± 2, 450 2 dakika boyunca x g ve aspire yüzer FACS tamponu santrifüj 100 ul ekle. </li><li> , Gerekli hücre yüzey markörü mAb seçin örneğin: CD45-BV421 (HI30, 1:20), CD3-ECD (UCHT1, 1:50), CD4-PC5.5 (1388,2, 1: 200), CD8-APC-AlexFluo700 (B9.11, 1: 400), CD14-FITC (TUK4, 1:50), CD19-APC-AlexFluo750 (13-119, 1:50), CD56-PE (MEM-188, 1:50), CD25-PeCy7 (BC96, 1:50), CD11c-PeCy7 (BU15, 1:10) ve CD1c-APC (AD5-8E7, 1:10), test edilen her mAb seyreltme faktörüne göre FACS tamponu kullanılarak mAb-karışım hazırlayın. olmayan leke numunesi ile birlikte antikor izotip kontrolü kullanarak kapı ayarlarını tanımlayın. </li><li> Her kuyuya hazırlanan mAb-karışımının 25 ul ekle. ışıktan korumak, 20 ° C ± 2 20 dakika kuluçkalayın. </li><li> 20 ° C ± 2, 450 2 dakika boyunca x g ve aspire yüzer FACS tamponu santrifüj 100 ul ekle. </li><li> Sadece hücre yüzey boyama gerektiren numuneler için, adım 3.16 ile devam edin. </li><li> hücre içi Foxp3 boyama durumunda: <ol><li> seyrelticiler 3 parça ile, konsantratın bir kısım karıştırılarak tespit ve permeabilizasyon tamponu hazırlayın. </li><li> 1x permeabilization tampon hazırlayın: sterilize H 2 O 1 kısım 10x permeabilization tamponu + 9 parça </li></ol></li><li> fiksasyon ve permeabili 100 ul pelet tekraryon tamponu, iyice karıştırın ve 4 ° C&#39;de inkübe 30 dakika ° C. </li><li> 450 2 dakika boyunca oda sıcaklığında xg, ve aspirat yüzer her şey, santrifüj 100 ul permeabilizasyon tamponu ekleyin. </li><li> permeabilizasyon tamponu bir kez daha hücreleri yıkayın. </li><li> Örnek için gerekli hücre içi mAb seçin, Foxp3-eFluo450 (PCH101, 1:50), IL-17A-AlexFluo488 (eBio64DEC1, 1:50) ve IFNy-PeCy7 (4S.B3, 1: 400). Test edilen her bir mAb seyreltme faktörüne göre% 2 normal sıçan serumu içeren permeabilizasyon tamponu kullanılarak mAb&#39;ler karışım hazırlayın. olmayan leke numunesi ile birlikte antikor izotip kontrolü kullanarak kapı ayarlarını tanımlayın. </li><li> Her kuyuya hazırlanan mAb karışımı 20 ul ekle. 4 inkübe ışıktan korumak, 30 dakika boyunca ° C. </li><li> 30 dakika inkübasyondan sonra, her kuyuya permeabilizasyon tampon 100 ul ilave edin. 20 ° C ± 2, 2 dakika boyunca 450 x g&#39;de santrifüj ve aspire yüzer. </li><li> Permeabilization Buffe hücreleri yıkayınr bir kez daha. microFACS tüpe 110 FACS tampon ul, ve transfer hücre süspansiyonları içinde süspanse pelet. NOT: Örnek 10 renkli akım sitometri kullanarak ölçüm için hazırdır. </li><li> Gerekli olan tam hücre sayısı halinde hemen akış sitometrisi kullanılarak ölçümler önce her numune içine florosferlerin iyi karıştırılmış düzgün süspansiyonu 10 ul ekle. </li></ol>

<ol>
	<li>Clark, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vast+majority+of+CLA++T+cells+are+resident+in+normal+skin.">The vast majority of CLA+ T cells are resident in normal skin.</a> J Immunol. 176, 4431-4439 (2006).</li><li>Zaba, L. C., Fuentes-Duculan, J., Steinman, R. M., Krueger, J. G., Lowes, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+dermis+contains+distinct+populations+of+CD11c+BDCA-1++dendritic+cells+and+CD163+FXIIIA++macrophages.">Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.</a> J Clin Invest. 117, 2517-2525 (2007).</li><li>Gebhardt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T+cells+in+nonlymphoid+tissue+that+provide+enhanced+local+immunity+during+infection+with+herpes+simplex+virus.">Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus.</a> Nature Immunol. 10, 524-530 (2009).</li><li>Boyman, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+development+of+psoriasis+in+a+new+animal+model+shows+an+essential+role+for+resident+T+cells+and+tumor+necrosis+factor-alpha.">Spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-alpha.</a> J Exp Med. 199, 731-736 (2004).</li><li>Christophers, E., Mrowietz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammatory+infiltrate+in+psoriasis.">The inflammatory infiltrate in psoriasis.</a> Clin Dermatol. 13, 131-135 (1995).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+infection+generates+non-migratory+memory+CD8++T(RM)+cells+providing+global+skin+immunity.">Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.</a> Nature. 483, 227-231 (2012).</li><li>Havran, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+diversity+of+T-cell+receptor+gamma-chain+expression+of+murine+Thy-1++dendritic+epidermal+cells+revealed+by+V+gamma+3-specific+monoclonal+antibody.">Limited diversity of T-cell receptor gamma-chain expression of murine Thy-1+ dendritic epidermal cells revealed by V gamma 3-specific monoclonal antibody.</a> Proc Natl Acad Sci U S A. 86, 4185-4189 (1989).</li><li>Campbell, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+expression+and+memory+T+cell+diversity+in+humans.">CCR7 expression and memory T cell diversity in humans.</a> J Immunol. 166, 877-884 (2001).</li><li>He, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+PKC+in+human+T+cells+using+sotrastaurin+(AEB071)+preserves+regulatory+T+cells+and+prevents+IL-17+production.">Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.</a> J Invest Dermatol. 134, 975-983 (2014).</li><li>Clark, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+isolation+of+skin+resident+T+cells+from+normal+and+diseased+human+skin.">A novel method for the isolation of skin resident T cells from normal and diseased human skin.</a> J Invest Dermatol. 126, 1059-1070 (2006).</li><li>Clark, R. A., Kupper, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+and+dermal+fibroblasts+induce+proliferation+of+natural+regulatory+T+cells+isolated+from+human+skin.">IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin.</a> Blood. 109, 194-202 (2007).</li><li>Keijsers, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+induction+of+cutaneous+inflammation+results+in+the+accumulation+of+extracellular+trap-forming+neutrophils+expressing+RORgammat+and+IL-17.">In vivo induction of cutaneous inflammation results in the accumulation of extracellular trap-forming neutrophils expressing RORgammat and IL-17.</a> J Invest Dermatol. 134, 1276-1284 (2014).</li><li>Hybbinette, S., Bostrom, M., Lindberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+dissociation+of+keratinocytes+from+human+skin+biopsies+for+in+vitro+cell+propagation.">Enzymatic dissociation of keratinocytes from human skin biopsies for in vitro cell propagation.</a> Experimental dermatology. 8, 30-38 (1999).</li><li>Hennino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-infiltrating+CD8++T+cells+initiate+atopic+dermatitis+lesions.">Skin-infiltrating CD8+ T cells initiate atopic dermatitis lesions.</a> J Immunol. 178, 5571-5577 (2007).</li></ol>

The authors have nothing to disclose.

Burada, verimli insan derisi biyopsi cilt yerleşik T hücrelerini izole etmek için bir protokol mevcut. Bu protokolün avantajı canlı lenfositlerinin nispeten yüksek sayılar izolasyon ve ilgili yüzey belirteçlerinin ifade. Belirlenen hücre alt edildi: CD11c + DH&#39;ler, CD4 + ve CD8 + T hücreleri ve Foxp3 + CD25 + hücreleri. Önemli olarak, izole edilmiş bir cilt yerleşik T hücrelerinin ex vivo kültür çok iyi uygulanabilir ve daha sonra fo...

cilt, vücut ve çevre arasındaki temel arayüz olarak, dış fiziksel, kimyasal ve yaralama, ultraviyole radyasyon ve mikro-organizmalar gibi biyolojik hakaretlerle karşı ilk savunma hattını sağlar. Cilt iki bölme, epidermis ve dermiş içerir ve Langerhans hücreleri, makrofajlar, dendritik hücreler (DC) ve yaklaşık 20 milyar bellek T hücreleri dahil olmak üzere bağışıklık hücreleri, çeşitli tüm kan hacmi 1 &#39;de yaklaşık iki katı sayıda mevcut içerir 2. Verilerin artan vücut deri doku homeostazında sırasında ve çeşitli patolojik durumlarda, hem temel immünolojik fonksiyonu vardır görüşünü desteklemektedir. Normal deride yerleşik bağışıklık hücreleri immüno 3 yapmak için düşünülen ve sedef 4 gibi iltihaplı bozuklukların gelişiminde bir rol oynadığı gösterilmiştir. Psöriatik lezyonlu deride, CD4 + ve CD8 + T hücrelerinin hem obs vardı sızmışerved ve CD4 ve CD8 oranı hastalığın durumu 5 bağlı olarak değişir gösterilmiştir. Bununla birlikte, hücrelerin bu popülasyonlar, mevcut tekniklerin sadece birkaç hücre yalıtılmasını için çalışma zordur. İnsan derisinden T hücre izolasyonu için şu anda yaygın olarak kullanılan teknikler enzimatik tedavi ile mekanik cilt ayrışmasını birleştirir. İnsan deri biyopsileri yoğun Tripsin, kolajenaz ve / veya EDTA 6-8 gibi enzimler ile inkübe edilir. Cilt germe kuvvetleri ve mekanik ayrıştırma yüksek dirençli bir bariyer doku olduğu göz önüne alındığında, T hücre izolasyon kurulan yöntemleri bu hücre popülasyonlarının zor ve ex vivo hücre kültürü yapar çok az hücre ve canlı hücrelerin daha düşük sayıda üretilen meydan okuma. Burada, mechan birleştirerek sağlıklı ve ilgili her iki psoriatik insan cildi lenfositlerin izole edilmesi için bir tadil edilmiş yöntem raporenzimatik sindirim kolajenaz ile birlikte otomatik bir doku Dissociator yerine yoğun kıyma kurulan yöntemi kullanarak deri iCal&#39;ın ayrışma. DH&#39;ler ve T hücrelerini içeren çeşitli canlı immün hücre alt tek bir hücre süspansiyonunun hazırlanması sonra görülmüştür. Önemli yüzey markörlerinin CD3, CD4 ve CD8 salgılanma iyi korunmuştur. Bu şekilde hazırlanan hücreler, ex vivo hücre kültürleri veya akış sitometrik analizinde kullanım için hazırdır. Bu protokol başarıyla sedef hastalarının lezyonlu deride türetilmiş tek cilt biyopsisi (4 mm) analizi için istihdam edilmiştir. Sonuçlar cilt ikamet hasta T hücreleri sağlıklı gönüllülere 9 karşılaştırıldığında IL-17 ve IFNy gibi daha inflamatuar sitokinler üretilen gösterdi.

<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

İnsan derisi önemli bir bariyer işlevi vardır ve patojenlerin doku homeostazında ve korunmasına katkıda çeşitli bağışıklık hücrelerini içerir. Cilt erişimi nispeten kolay olduğundan, periferik immün düzenleyici mekanizmaları incelemek için ideal bir platform sağlar. aynı zamanda bağışıklık yerleşik sağlıklı deri Davranış immüno hücrelerin ancak, sedef hastalığı gibi iltihaplı deri hastalıklarının gelişiminde önemli bir rol oynar. Gelişmekte olan anlayışlar rağmen, çeşitli iltihaplı cilt hastalıkları altında yatan biyolojinin anlayışımız hala sınırlıdır. biyopsi yapılan deri numunelerinden izole kaliteli (tek) hücre popülasyonları için bir ihtiyaç vardır. Şimdiye kadar, izolasyon prosedürleri ciddi canlı hücre yeterli sayıda elde eksikliği ile engellenmiştir edilmiştir. İzolasyon ve sonraki analizler nedeniyle de elde etmek için geçerli ayrılma işlemleri nedeniyle mekanik ve kimyasal strese, immün hücre soyu belirteçleri kaybı etkilenmişTek hücre süspansiyonu. Burada, otomatik bir doku aynştıncı ve kollajenaz muamelesi ile mekanik deri ayrışmasını birleştirerek sağlıklı ve ilgili her iki psoriatik insan deri T hücrelerinin izole edilmesi için bir tadil edilmiş yöntem açıklanmaktadır. Bu yöntem, tek hücre süspansiyonlarının hazırlanması üzerine, CD4, CD8, Foxp3 ve CD11c gibi en bağışıklık soy işaretlerinin ifadesi korur. Başarılı CD4 + T hücre izolasyonu ve sonraki fenotipik ve fonksiyonel analiz örnekleri gösterilmiştir.

Burada sunulan protokol, tek bir 4 mm deri biyopsisi kullanırken 760 ± 178.000 kadar 615 (± SEM, sağlıklı gönüllülerin cilt demek) ± 2.200 arasında insan derisinden canlı lenfositler (± SEM, sedef hastalarının lezyonlu deriye ortalama) verecektir. CD45 + hücrelerinin farklı CD4 + T hücreleri (~% 45), CD8 + T hücreleri (% 30) da dahil olmak üzere, sağlıklı bireylerin derisinden türetilen tek hücre süspansiyonları olarak tanımlanır ...

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.

Fenotipik Analizi ve İnsan Cilt lenfosit izolasyonu<em&gt; Ex Vivo</em&gt; Hücre Kültürü

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 Görüntüleme.  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.

Video: 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.

Göç İnsan T Lenfosit İzolasyon, Kültür ve Analiz In Vitro</em

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

İmmünoloji ve Enfeksiyon

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.

Floresan-aktif hücre sıralama ile ınsan derisinden papiller ve Reiküler fibroblastların izolasyonu

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

Genetik

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Birincil insan Lenfositi yetişkin cilt dokusundan izole etmek için basit ve etkili bir yöntem

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Gelişim Biyolojisi

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

İnfiltratif Lenfositlerinin Analiz Olmayan enzimatik ayırmak Taze İnsan Dokusuna Basit ve Hızlı Protokolü

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

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.

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.

Farklı Fare Cilt Bölgelerinden sitoflüorimetrik analize için tek hücre süspansiyonları hazırlanması

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

Enzimatik Digest ve Gradient Ayırma kullanma Fare Skin gelen sızan lökositlerin İzolasyonu

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

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Canlı zayıflatılmış intradermal aşılama sonrasında Fare Deri ve akan lenf düğümü gelen Myeloid Hücre İzolasyonu<em&gt; Plasmodium</em&gt; Sporozoitler

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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.

Çoklu-parametre floresans ile aktive edilen hücre sınıflandırma İnsan lenfatik endotel hücreleri İzolasyonu

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

Tıp

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.

Lenfositler fare küçük bağırsak bağışıklık sistemi yalıtma

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

Murin Dermal Lenfatik Kılcal Damarlardan Endotel Hücrelerinin İzole Edilmesi

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Yazar Spotlight: Murin Dermal Lenfatik Endotel Hücrelerinin Manyetik Boncuk Tabanlı İzolasyonu

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Murin Dermal Lenfatik Endotel Hücre İzolasyonu

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...

Fenotipik Analizi ve İnsan Cilt lenfosit izolasyonu

Özet

Daha Fazla Video Keşfet

Göç İnsan T Lenfosit İzolasyon, Kültür ve Analiz In Vitro