biópsias de pele de pacientes com psoríase foram gentilmente cedidas pelo Dr. Andreas Koerber (departamento de Dermatologia da Universidade de Essen, Alemanha), após consentimento informado oral ou escrita para uso científico. XH também é suportado pelo NSFC 61263039 e 11101321 NSFC.

NOTA: As biópsias de pele de indivíduos saudáveis ​​foram obtidos a partir de restos de pele abdominal dos indivíduos submetidos a cirurgia plástica eletiva, após consentimento informado oral ou escrita para uso científico. O uso de pele humana foi aprovado e em conformidade com os regulamentos estabelecidos pelos médicos Comitês de Ética em Pesquisa com Seres Humanos centro médico da Universidade Radboud, Nijmegen, Holanda e Universidade de Essen, Alemanha. 1. Preparação de suspensões de células individuais de Pele Humana (Trabalho estéril em um Fluxo de-gabinete se Cultura de Células posterior é necessária) <ol><li> Prepare meio de cultura celular: RPMI 1640 + penicilina / estreptomicina (concentrações finais de 100 unidades / mL e 100 ug / ml, respectivamente) + piruvato (0.02 mM) e Glutamax (0,02 mM), sem soro adicionado. </li><li> Prepare meio de cultura completo: meio de cultura preparado no passo 1.1 + 10% de soro humano (HPS); Armazenar a 4 ° C. Traga médio a 20 ° C ± 2 antes de nósing. </li><li> Obter a biópsia de pele com um instrumento redondo 4 milímetros biópsia por punção e mantê-lo no meio de cultura RPMI 1640 completo a 20 ° C ± 2 até 4 horas ou a 4 ° C EM. Processar a biópsia, o mais rapidamente possível após a chegada ao laboratório. NOTA: um armazenamento mais prolongado da pele vai influenciar o rendimento das células e viabilidade celular. </li><li> Marcar um tubo de dissociação azul-tampado e adicionar 5 ml de meio de cultura completo para o tubo rotulado. </li><li> Adicionar 2 ml de meio de cultura completo a cada poço (num total de 3 cavidades) de uma placa de cultura de 6 poços estéril. Use ferramentas estéreis para colocar a biópsia em um único poço, lavar, mova-o para um segundo poço e repetir este passo mais uma vez, conseguindo assim um total de três lavagens. </li><li> Transferir a biópsia da pele bem lavado com uma placa de Petri estéril, adicionar 100 uL de meio de cultura completo no topo da biópsia, e raspar cuidadosamente o tecido gordo subcutâneo utilizando-se um bisturi de aço inoxidável estéril descartável. NOTA: Thé é um passo crítico. </li><li> Corte cada biópsia da pele em 4 pedaços menores em uma placa de Petri estéril. amostras de transferência (até quatro biópsias de 4 mm por tubo) para o tubo de dissociação preparadas contendo 5 ml de meio de cultura completo. </li><li> Feche bem os tubos com a tampa, e anexar cabeça para baixo para a manga da Dissociator tecido automatizado. Certifique-se de que todo o material de amostra está localizado na área do rotor. </li><li> Iniciar o processo de dissociação, executando o &quot;_01 programa m_spleen&quot; (um programa pré-definido fornecida pela memória interna do instrumento ou pelo cartão de programa, acompanhado) dissociar a biópsia na velocidade de rotação apropriada para 56 seg. </li><li> Após o processamento, retire o tubo de dissociação do Dissociator e certifique-se que todo o material dissociado é recolhido na parte inferior do tubo. </li><li> Adicionar 150 ul de colagenase IA (80 mg / ml) para dentro do tubo de dissociação e incubar a amostra num banho de água com agitação a 37° C durante 60 min. Adicionar 100 ul de ADNase I (5 mU / ml) para dentro do tubo thedissociation, misturar bem. NOTA: Maior concentração de colagenase ou tempo de incubação vai alterar a viabilidade celular. </li><li> Fixe o tubo de dissociação para a manga da Dissociator tecido automatizada e execute o programa &quot;m_ baço _01&quot; dissociar a biópsia mais uma vez. </li><li> Coloque um coador de células de nylon de 70 | iM no topo de um tubo Falcon de 50 ml. Aplicar materiais de amostra dissociados para este filtro celular para remover os detritos aglomerados de células / tecido. </li><li> Lavar uma vez com filtro de células 5 ml de meio de cultura completo. Centrifuga-se a 20 ° C ± 2, 450 xg durante 10 min e o sobrenadante aspirado. </li><li> Repetir o passo de lavagem mais uma vez. Ressuspender as peletes de células em 300 ul de meio de cultura completo. uma única célula suspensões estão prontos para uma análise mais aprofundada; continuar com protocolos para a cultura de células ex vivo ou citometria de fluxo. </li><li> Em caso de mais icoloração de citocinas ntracellular, estimular as células com PMA (12,5 ng / ml), ionomicina (500 ng / ml) e Brefeldine A (5? g / ml) durante 4 hr a 37 ° C, 5% de CO2 durante 4 horas antes de executar o fluxo análise de citometria. </li></ol> 2. Polyclonal ativação das células T da Pele-residente (Ex Vivo de Cultura Celular) <ol><li> Alíquotas de suspensões de células únicas de 100 ul (preparado no passo 1.15) numa placa de fundo redondo de 96 poços. NOTA: Para cada biópsia de pele de 4 mm, as suspensões unicelulares adquiridos pode ser dividida em, pelo menos, duas cavidades de uma placa de 96 poços. </li><li> Adicionar anti-CD3 / microesferas anti-CD28 mAb-revestidas (25.000 grânulos por poço), citocina recombinante humano de rIL-2 (concentração final de 25 U / ml) e rIL-1p (concentração final de 50 ng / ml). </li><li> Cobrir a placa de cultura com uma tampa de placa bem marcado, incubar em 5% de CO2, 100% de humidade, 37 ° C incubadora. Alterar médio quando a cor média vira para amarelo. <br/&gt; NOTA: Uma colônia de células claras pode ser observado a partir do dia 8 de cultura de células. </li></ol> 3. citometria de fluxo de primário / cultivadas células da pele Residente T <ol><li> Preparar tampão de FACS: PBS + 0,2% de BSA. </li><li> Transferir células de interesse em uma placa de 96 poços de fundo em V. Centrifuga-se a 20 ° C ± 2, 450 x g durante 2 min, e o sobrenadante aspirado. </li><li> Ressuspender o sedimento em 100 ul de PBS. Centrifuga-se a 20 ° C ± 2, 450 x g durante 2 min, e o sobrenadante aspirado. </li><li> As células são coradas com 100 ul de eFluorescence780 preparados conjugados corante de viabilidade fixável (1: 1000 de diluição utilizando PBS) a 4 ° C durante 30 min. Adicionar 100 ul de tampão de FACS, centrifugar a 20 ° C ± 2, 450 x g durante 2 min, e o sobrenadante aspirado. </li><li> Escolha mAbs marcador da superfície celular necessárias, por exemplo: CD45-BV421 (HI30, 01:20), CD3-ECD (UCHT1, 01:50), CD4-PC5.5 (1388,2, 1: 200), CD8-APC-AlexFluo700 (B9.11, 1: 400), CD14-FITC (TUK4, 01:50), CD19-APC-AlexFluo750 (13-1191:50), CD56-PE (MEM-188, 01:50), CD25-PeCy7 (BC96, 01:50), CD11c-PeCy7 (BU15, 1:10), e CD1c-APC (AD5-8E7, 1:10), preparar os mAbs-mistura, utilizando tampão FACS de acordo com cada fator de diluição mAb testados. Definir as configurações de porta usando o controle isotipo do anticorpo em conjunto com amostra não-mancha. </li><li> Adicionar 25 uL de mAb preparado-mistura em cada poço. Incubar 20 min a 20 ° C ± 2, proteger da luz. </li><li> Adicionar 100 ul de tampão de FACS, centrifugar a 20 ° C ± 2, 450 x g durante 2 min, e o sobrenadante aspirado. </li><li> Para amostras que exigem apenas coloração da superfície celular, continue com o passo 3.16. </li><li> No caso da coloração de Foxp3 intra-celular: <ol><li> Preparar tampão de fixação e permeabilização através da mistura de uma parte do concentrado com 3 partes de diluentes. </li><li> Prepare tampão de permeabilização 1x: 1 parte de 10x permeabilização tampão + 9 partes de H 2 O. esterilizado </li></ol></li><li> Ressuspender as pelotas em 100 ul de fixação e permeabilitampão zação, misture bem, e incubar a 4 ° C durante 30 min. </li><li> Adicionar 100 ul de tampão de permeabilização a cada poço, centrifugar a 450 xg à temperatura ambiente durante 2 min, e o sobrenadante aspirado. </li><li> Lavar as células com tampão de permeabilização mais uma vez. </li><li> Escolha mAbs intracelulares necessário, por exemplo, Foxp3-eFluo450 (PCH101, 01:50), IL-17A-AlexFluo488 (eBio64DEC1, 01:50) e de IFN-y-PeCy7 (4S.B3, 1: 400). Prepare a mistura de mAbs utilizando tampão de permeabilização contendo soro normal de rato 2% de acordo com cada fator de diluição mAb testados. Definir as configurações de porta usando o controle isotipo do anticorpo em conjunto com amostra não-mancha. </li><li> Adicionar 20 uL de mAb preparado-mistura em cada poço. Incubar a 4 ° C durante 30 min, proteger da luz. </li><li> Após 30 min de incubação, adicionar 100 ul de tampão de permeabilização em cada poço. Centrifuga-se a 20 ° C ± 2, 450 x g durante 2 min, e o sobrenadante aspirado. </li><li> Lave as células com buffe permeabilizaçãoR mais uma vez. Ressuspender sedimento em 110 ul de tampão de FACS, e suspensões de células para dentro do tubo de transferência microFACS. NOTA: A amostra está pronta para a medição usando 10 cores citometria de fluxo. </li><li> No caso do número de células exato necessário, adicione 10 ml de suspensão uniforme bem misturado de fluoroesferas em cada amostra imediatamente antes de realizar medições de citometria de fluxo. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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The authors have nothing to disclose.

Aqui, apresentamos um protocolo para isolar eficientemente células T residentes pele de biópsias de pele humana. A vantagem deste protocolo é o isolamento de um número relativamente elevado de linfócitos viáveis, e expressando marcadores de superfície relevante. As subpopulações de células identificadas foram: células CD11c + DCs, CD4 + e CD8 + T e Foxp3 + CD25 +. Importante, a cultura ex vivo de células T da pele isolado residentes foi muito be...

A pele, como a interface primária entre o corpo e o ambiente, proporciona a primeira linha de defesa contra físico externo, insultos químicos e biológicos, tais como ferimentos, radiação ultravioleta e micro-organismos. Pele compreende dois compartimentos principais, a epiderme e a derme, e contém uma variedade de células imunitárias, incluindo células de Langerhans, macrófagos, células dendríticas (DC), e cerca de 20 mil milhões de células T de memória, quase o dobro do número presente em todo o volume de sangue 1 , 2. Um crescente corpo de dados suporta a noção de que a pele tem funções imunológicas essenciais, tanto durante a homeostase do tecido e em várias condições patológicas. Células imunes residentes na pele normal são pensados ​​para conduzir imunovigilância e 3 têm demonstrado desempenhar um papel no desenvolvimento de doenças inflamatórias tais como psoríase 4. Na pele lesionada psoriática, tanto CD4 + e CD8 + foram infiltradas células T observed e foi demonstrado que a razão entre a CD4 e CD8 varia dependendo do estado da doença 5. No entanto, estas populações de células são difíceis de estudar porque as técnicas existentes permitem o isolamento de apenas algumas células. As técnicas atualmente utilizados para o isolamento de células T a partir de pele humana combinam dissociação da pele mecânica com tratamento enzimático. As biópsias da pele humana são extensamente triturada e incubada com enzimas como a tripsina, colagenase e / ou EDTA 6-8. Considerando-se que a pele é um tecido de barreira, que é altamente resistente a forças de tracção e de desagregação mecânica, os métodos estabelecidos de isolamento de células T produzido muito poucas células, e até mesmo menor número de células viáveis, o que torna a cultura ex vivo de células destas populações de células difíceis e desafiador. Aqui, nós relatamos um método modificado para isolar linfócitos de pele humana psoriática saudável e envolvido pela combinação mechanical dissociação da pele usando um Dissociator tecido automatizada em vez do método estabelecido de picagem extensivamente, em conjunto com a digestão enzimática com colagenase. Vários subconjuntos de células imunitárias, incluindo as DCs viáveis ​​e células T foram observadas após a preparação de uma suspensão de célula única. É importante notar a expressão dos marcadores de superfície CD3, CD4 e CD8 foi bem preservada. As células assim preparadas, estão prontos para uso em culturas de células ex vivo ou análise de citometria de fluxo. Este protocolo tem sido empregue com sucesso para a análise de biópsias de pele individuais (4 mm) derivadas a partir da pele lesionada de pacientes com psoríase. Os resultados mostraram que as células T do paciente de pele residente produzido mais citocinas inflamatórias como a IL-17 e IFN-y, em comparação com voluntários saudáveis ​​9.

<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

A pele humana tem uma função de barreira importante e contém várias células imunitárias que contribuem para a homeostase dos tecidos e protecção contra agentes patogénicos. Como a pele é relativamente fácil de acesso, ele fornece uma plataforma ideal para estudar mecanismos de regulação do sistema imunológico periféricos. células residentes na conduta imunes pele saudável imunovigilância, mas também desempenhar um papel importante no desenvolvimento de doenças de pele inflamatórias, tais como a psoríase. Apesar percepções emergentes, a nossa compreensão da biologia subjacente várias doenças de pele inflamatória é ainda limitada. Há uma necessidade de populações de células (single) de boa qualidade isoladas de amostras de pele biopsiados. Até agora, os procedimentos de isolamento ter sido seriamente dificultados pela falta de obtenção de um número suficiente de células viáveis. Isolamento e análise posterior também foram afetados pela perda de marcadores de linhagem de células do sistema imunológico, devido ao estresse mecânico e químico causado pelos procedimentos de dissociação atuais para obtersuspensão de células individuais. Aqui, descrevemos um método modificado para isolar as células T a partir de pele humana psoriática saudável e envolvido pela combinação de dissociação da pele mecânica usando um Dissociator de tecido automático e tratamento de colagenase. Esta metodologia preserva expressão da maior parte dos marcadores de linhagem imunes, tais como CD4, CD8, CD11c e Foxp3 mediante a preparação de suspensões de células únicas. Os exemplos de sucesso de isolamento de células T CD4 + e fenotípica subsequente e análise funcional são mostrados.

O protocolo apresentado aqui vai render entre 2.200 ± 615 (média ± SEM, pele de voluntários saudáveis) até 178.000 ± 760 (média ± SEM, pele lesionada de pacientes com psoríase) linfócitos viáveis ​​de pele humana quando se utiliza uma única biópsia de pele de 4 mm. Foram identificados diferentes tipos de CD45 + células em suspensões unicelulares derivadas da pele de indivíduos saudáveis, incluindo as células T CD4 + (~ 45%), as células T CD8 <sup...

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.

Isolamento de linfócitos da pele humana para análise fenotípica e<em&gt; Ex Vivo</em&gt; Cultura de Células

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 visualizações.  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.

Vídeo: 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.

Isolamento T de linfócitos humanos, Cultura e Análise da Migração 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 ...

Imunologia e Infecção

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.

Isolamento de fibroblastos papilares e reticular da pele humana por triagem celular ativada por fluorescência

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

Genética

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.

Um método simplificado e eficiente para isolar os queratinócitos humanos primários do tecido da pele de adultos

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

Biologia do Desenvolvimento

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.

Um protocolo simples e rápido para não enzimática Separar frescos Tecidos Humanos para a Análise de infiltrar Linfócitos

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.

Preparação de uma única célula suspensões para Análise de citometria de fluxo de diferentes regiões da pele do rato

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.

Isolamento de leucócitos de infiltração de rato pele utilizando digestão enzimática e separação por gradiente

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.

Isolamento de células mielóides de rato Pele e Drenagem Linfonodo Seguindo intradérmica imunização com vivos atenuados<em&gt; Plasmodium</em&gt; Esporozoítos

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.

Isolamento de linfático humano endotelial por multi-parâmetro activadas por fluorescência celular

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

Medicina

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.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

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

Isolando células endoteliais de capilares linfáticos dérmicos murinos

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.

Autor em destaque: Isolamento baseado em esferas magnéticas de células endoteliais linfáticas dérmicas murinas

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.

Isolamento de células endoteliais linfáticas dérmicas murinas

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

Isolamento de linfócitos da pele humana para análise fenotípica e

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Isolamento T de linfócitos humanos, Cultura e Análise da Migração In Vitro