Os autores reconhecem o apoio por concessões do Deutsche Forschungsgemeinschaft SI-1281/4-1 e BIOS da Faculdade de Medicina da Universidade de Freiburg (para CS).

1. Preparação de Anticorpo Pathogenic Nota: A purificação e concentração dos anticorpos deve ser realizada no mesmo dia, na forma de anticorpos não são para ser armazenada em glicina 0,1 M pH 2,5-3 (tampão de eluição) durante a noite (ON). A purificação por afinidade de IgG: usar 25 ml de soro de coelho imune: <ol><li> Descongelar o soro de coelho ON a 4 ° C, mistura-a 1:1 com tampão de corrida (1xPBS) e centrifugar a 1260 ela xg durante 10 min a 20 ° C. Opcionalmente, um passo de filtração com papel de filtro pode ser incluída no processo, após a centrifugação do soro é gordurosa. </li><li> Enquanto isso, lava-se a proteína G matriz cheia de coluna com 10 volumes de leito (volume do leito = volume da matriz) de 1xPBS (tampão de corrida). </li><li> Aplicar o soro diluído e filtrou-se para a coluna e incuba-se 1 hora na plataforma de balanço à temperatura ambiente (RT). </li><li> Recolher o flow-through (FT) num tubo de Flacon de 50 ml. Não descartá-lo até a concentraçãoe titulação da IgG purificada é conhecido. </li><li> Lava-se a matriz com 5 volumes de leito de 1xPBS e, entretanto, se preparar tubos de 50 ml Falcon para recolher a fracção de IgG eluida. Pipetar 1 ml de tampão de neutralização de pH: Tris 1 M pH 10, em cada um dos tubos (caso contrário, a proporção de 01:20 de tampão TRIS em relação à quantidade de fluido a ser recolhido). </li><li> Aplicar 100-150 ml de tampão de eluição: glicina 0,1 M pH 2,5-3 para a coluna e recolher 50 ml fracção (s). Virar o tubo 2-3x, medir o pH e adicionar tampão de neutralização até que tenha atingido o pH 7,2-7,4. </li><li> Recolher gotas de fluido e IgG utilizando o tampão de eluição, como etalon medir a densidade óptica a 280 nm. Se a DO for &lt;recolha de paragem 0,1. </li><li> Regenerar a proteína da matriz G e armazená-la seguindo as instruções do fabricante. </li></ol> Concentração por ultrafiltração Amicon com tubos: <ol><li> Lavar o Ultra Amicon (15 ml / 30 kDa) em tubos de ultraf iltração com 1xPBS por centrifugação 15-20 minem 3220 xg, a 4 ° C. Descarte o FT do Amicon. </li><li> Encha as Amicons com 15 ml da fracção de IgG eluida e centrifugar durante 25-30 minutos a 3220 xg e 4 ° C, o tempo de centrifugação sempre depende do teor de proteína do eluato. </li><li> Descarte FT e repetir ultrafiltração até que você tem nenhuma fração eluída esquerda. </li><li> Lava-se a IgG concentrado extensivamente, por duas vezes, com 1xPBS 25-30 min por centrifugação. </li><li> Recolher o &quot;adesivo&quot;, a solução de anticorpo amarelado a partir do filtro Amicon, num volume final de 1.500-2.000 ul de 1xPBS. </li><li> Concentração medida de preparação de IgG utilizando um espectrofotómetro ou NanoDrop: Conc. (Mg / ml) = A (280 nm) x factor de diluição / 1,4 </li><li> Lavar e armazenar os Amicons seguindo as instruções do fabricante. </li></ol> 2. A injeção de colágeno VII IgG específica em camundongos Antes de iniciar o procedimento real experimental, certifique-se de que o pró experimentalprotocolo é escrito e todos os materiais são preparados para o experimento. <ol><li> Prepare as folhas de dados para o placar doença, ELISA, imunofluorescência (IF) análise e mieloperoxidase (MPO) ensaio 4. </li><li> Rotular os tubos de sangue e órgãos, cryomolds bem como histologia de processamento e incorporação de cassetes. </li><li> As soluções de anticorpo deve ser preparado fresco ou descongelado a partir de stocks e caracterizado quanto à sua reactividade (título por microscopia de IF e / ou ELISA). A solução filtrada estéril anticorpo deve ser diluído em PBS, de forma que a dose necessária para a injecção varia entre 250-1000 ul. Certifique-se de que você tem IgG suficiente para realizar todo o experimento. </li><li> Preparar fresco anticoagulante heparina de 20 U / ml e 8,7 mg / ml de cetamina / xilazina 1,3 mistura mg / ml para a anestesia. Quando sacrificar os ratos têm rotulado tubos com formaldeído 3,7% preparado. </li><li> Esterilizar instrumentos cirúrgicos (bisturi, tesouras, ponta fina e Curvforcipes ed) e preparar seringas (seringas de insulina, 1 e 2 seringas ml), agulhas, câmera digital e cartão de memória extra. </li></ol> Nota: Execute todos os procedimentos de cetamina / xilazina ou isoflurano ratos narcotizada isoflurano é a opção preferida para anestesia as verificações diárias de pele, como os ratos recuperar rapidamente.. No entanto, ao tirar fotos, 87 cetamina mg / kg e xilazina 13 mg / kg é geralmente administrado por via subcutânea. Para a eutanásia a dosagem de cetamina / xilazina é aumentada para 130 mg de cetamina / kg de xilazina e 20 mg / kg. <ol><li> Verifique os animais já marcados para a sua saúde da pele estado geral, e da aparência da pele, pesá-los e medir a sua espessura orelha (sempre manter a mesma orelha). </li><li> Registre-se valores observados e mudanças na folha de avaliação clínico. </li><li> Recolher 20-30 ul (2-3 gotas) de sangue a partir da veia da cauda de uma seringa contendo o mesmo volume de anticoagulante. </li><li> Desinfectar a pele eda pele utilizando etanol a 80%. </li><li> Injectar a solução de anticorpo por via subcutânea na parte de trás utilizando uma seringa de insulina. (Para certos sítios (por exemplo, as orelhas) apenas pequenos volumes (10-50 ul) são viáveis ​​para ser injectada.) </li></ol> As injeções devem ser repetidas a cada dois dias, quatro vezes. Balb / c e ratinhos C57BL6 de um peso corporal de aproximadamente 20 g de começar a desenvolver a doença de 3-4 dias após a primeira administração de 400-500 até 750 ug / g de peso corporal / injecção auto-anticorpos. Ao terminar a experiência: <ol><li> Colocar os ratos sob narcose profunda por injecção de 130 mg / kg e 20 mg / kg de cetamina / xilazina por via subcutânea, desangrar los da veia cava posterior e cortar o coração. </li><li> Coletar amostras de tecidos / órgãos como a pele: rabo, orelha, sangue e esôfago e colocá-los em tubos previamente identificados e PBS ou 3,7% de formaldeído preenchido. </li></ol> Ao retornar para olaboratório: <ol><li> Centrifuga-se o sangue a 1200 xg, 10 min à temperatura ambiente para separar o plasma, transferir o plasma para tubos adequadamente etiquetados e armazená-los em conformidade. </li><li> Incorporar o biópsia da pele em meio corte de óptima temperatura através cryomolds, devidamente etiqueta, e armazená-los a -80 ° C. </li><li> Coloque as amostras de tecidos destinados a histologia nos moldes de incorporação rotulados e mantê-los em formol 3,7% até parafina. </li></ol> 3. Avaliação clínica de gravidade da doença Ratos devem ser examinados diariamente para lesões de pele e mucosa e os resultados devem ser registradas utilizando formulários apropriados. Os critérios para a eutanásia precoce é lesões da pele que afectam mais do que 20% da superfície do corpo e uma perda de peso de 5-10% do peso corporal total, em três dias consecutivos, contados a doença para uma pontuação de 5 (Tabela 1). <ol><li> Posicione o mouse sobre sua barriga. Comece com a área da cabeça, ou seja, com o direitoorelha e verificar se há bolhas, erosões, crosta no interior, bem como sobre o lado exterior. Continuar da mesma maneira com a orelha esquerda. </li><li> Olhos direito e esquerdo são controlados por sinais de eritema, alopecia, erosões e / ou crosta. </li><li> Testa e área focinho são escovados através seguinte, continuando com a parte ventral do focinho, lábio e da mucosa da boca. </li><li> Examine a perna dianteira direita a partir da pata, movendo-se para cima; mudar para a perna dianteira esquerda. Mesmo procedimento com as pernas traseiras direita e esquerda. </li><li> Pincele com a pele no pescoço e nas costas com uma área de curvas pinça de ponta fina. </li><li> Vire o mouse em sua parte traseira e verifique a parte ventral, bem como nos lados ventral dos membros da mesma forma que anteriormente. </li><li> Verifique a cauda. </li><li> Tire fotos de partes relevantes do corpo onde os ratos desenvolvem a doença. Atenção deve ser dada à qualidade e quantidade de fotos. </li><li> Calcular as pontuações clínicos da doença. </li></ol> 4. Análise de amostras de pele e Plasma <ol><li> Preparar, armazenar e / ou processar todos os tecidos coletados de acordo com o plano. </li><li> Corte cortes de tecidos congelados para se a detecção de anticorpos de coelho e rato componentes do sistema complemento. </li><li> Analisar o teor de proteína e de enzima diferente (ensaio de MPO) de tecidos congelados e / ou extractos de órgãos. </li><li> Secção do tecido embebido em parafina e coradas com H &amp; E para visualizar a extensão do dano tecidual. </li><li> Analisar o plasma por ELISA, imunotransferência. </li></ol>

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A transferência passiva de anticorpos em animais experimentais é uma abordagem importante para a demonstração da sua patogenicidade 7, -12. Os modelos animais obtidos por meio deste método, para além de ser a evidência indirecta para autoimunidade 13, permite a investigação da fase eferente do mecanismo patogénico. O modelo de transferência passiva de anticorpos induzida pela pele de granulócitos-dependente de bolhas Epidermólise bolhosa adquirida (EBA), foi usada para dissecar os meca...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> antigénio recombinante </td><td></td><td></td><td> A codificação de plasmídeos para as formas recombinantes do colagénio murino VII estão disponíveis a partir do autor correspondente. </td></tr><tr><td> soro de coelho imune </td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td> Tivemos coelhos New Zealand White imunizados com 200 ug de antigénio, 3 vezes em 2 semanas de intervalo. Para isso, usaram os serviços de Eurogentec SA, Bélgica. </td></tr><tr><td> Proteína G agarose </td><td> Roche Applied Science </td><td> 11243233001 </td><td></td></tr><tr><td> Camundongos Balb / c </td><td> Charles Ri ver Laboratories </td><td></td><td></td&gt; </tr><tr><td> Outubro composto, Tissue-Tek </td><td> Sakura Finetek </td><td> 4583 </td><td> OCT, a temperatura de corte óptima </td></tr><tr><td> Cryomold padrão </td><td> Sakura Finetek </td><td> 4557 </td><td> 25 milímetros × 20 mm × 5 mm </td></tr><tr><td> Cryomold intermediário </td><td> Sakura Finetek </td><td> 4566 </td><td> 15 mm × 15 mm × 5 mm </td></tr><tr><td> Uni-Link Einbettkassette- </td><td> R. Langenbrinck </td><td> 09-0503 </td><td> Histologia processamento e cassetes de incorporação </td></tr><tr><td> Spezial-Tatowierfarbe Schwarz </td><td> H. Hauptnerund &amp; Richard Herberholz GmbH &amp; Co. KG </td><td> 71492000 </td><td> tatuagem pasta </td></tr><tr><td> Tatowierzange TZ1 </td><td> EBECO E. Becker &amp; Co GmbH </td><td></td><td> tatuagem dispositivo </td></tr><tr><td> Heparina </td><td> Carl Roth GmbH &amp; Co. </td><td> 7692,1 </td><td></td></tr><tr><td> De formaldeído a 37% </td><td> Carl Roth GmbH &amp; Co. </td><td> 7386 </td><td></td></tr><tr><td> Cloridrato de cetamina </td><td> Sigma-Aldrich Chemie GmbH </td><td> K2753-1G </td><td></td></tr><tr><td> Cloridrato de xilazina </td><td> Sigma-Aldrich Chemie GmbH </td><td> X1251-1G </td><td></td></tr><tr><td> PBS estéril </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td> câmera digital </td><td> Nikon </td><td> Coolpix 5400 </td><td></td></tr><tr><td> Seringa conduzida unidade de filtro de 0,45 um </td><td> Millipore </td><td></td><td></td></tr><tr><td> Compasso de calibre </td><td> Mitutoyo 7309 </td><td> 1667338 </td><td> Farnell (distribuidor) </td></tr><tr><td> folha de pontuação doença </td><td></td><td></td><td> exemplo recintosed </td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

Fenómenos auto-imunes ocorrem em indivíduos saudáveis, mas, quando a auto-tolerância falhar, a resposta auto-imune pode resultar na patologia específica. De acordo com os postulados de Witebsky, um dos critérios para o diagnóstico de uma doença auto-imune, como seja a reprodução da doença em animais experimentais por uma transferência passiva de anticorpos. Para epidermólise bolhosa adquirida (EBA), uma doença órgão-específica protótipo auto-imune da pele e membranas mucosas, vários modelos experimentais foram recentemente estabelecida. No modelo animal descrito no nosso trabalho, purificou anticorpos IgG contra uma extensão de 200 aminoácidos (aa 757-967) de colagénio VII são injectados repetidamente em ratinhos que reproduzem o fenótipo empolar, bem como a histo-e imunopatológicas características de humano EBA 1. Full-blown doença generalizada é normalmente visto 5-6 dias após a primeira injecção e a extensão da doença está correlacionada com a dose administrada de a collagen VII-IgG específico. A lesão tecidual (a formação de bolhas) na EBA experimental é, dependendo do recrutamento e ativação de granulócitos por tecidos ligados auto-anticorpos 2, -4. Portanto, este modelo permite a dissecção da via dependente de granulócitos-inflamatório envolvido no dano de tecido auto-imune, como o modelo reproduz apenas a fase de células T independente da resposta auto-imune eferente. Além disso, o seu valor é sublinhado por uma série de estudos que demonstram o potencial de indução de blister de anticorpos in vivo e investiga o mecanismo da formação de bolhas na EBA 1,3, -6. Finalmente, o modelo vai facilitar grandemente o desenvolvimento de novas terapias anti-inflamatórios em doenças induzidas por auto-anticorpos. No geral, o modelo animal passiva transferência da EBA é um modelo de doença acessível e instrutivo e vai ajudar os pesquisadores a analisar não só a patogênese EBA, mas a resposta fundamental biológica e clinicamenteperguntas essenciais autoimunidade.

A transferência passiva de anticorpos específicos de antigénio resulta numa doença soprado em ratinhos, assemelhando-se a níveis clínicos, histológicos e imunopatológicas, a ABE humano. Bolhas, crostas, erosões e alopecia desenvolver nas orelhas, focinho, patas, pernas, costas e ao redor dos olhos dos camundongos. Os primeiros sinais clínicos da doença, muito provavelmente aparecer nas orelhas e / ou área de cabeça. Deposição de coelho específico IgG e complemento do mouse C3 é detectado pelo IF direta ...

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Granulocyte-dependent Autoantibody-induced Skin Blistering

No modelo animal descrito no nosso trabalho, purificou anticorpos IgG contra uma extensão de 200 aminoácidos (aa 757-967) de colagénio VII são injectados repetidamente em ratinhos que reproduzem o fenótipo empolar, bem como a histo-e imunopatológicas características de humano epidermólise bolhosa adquirida (EBA)<sup&gt; 1</sup&gt;.

Granulócitos-dependente Blistering pele induzida por auto-anticorpos

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

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JoVE Journal

Immunology and Infection

10.4K visualizações.  University of Freiburg. No modelo animal descrito no nosso trabalho, purificou anticorpos IgG contra uma extensão de 200 aminoácidos (aa 757-967) de colagénio VII são injectados repetidamente em ratinhos que reproduzem o fenótipo empolar, bem como a histo-e imunopatológicas características de humano epidermólise bolhosa adquirida (EBA) 1.

Vídeo: Granulocyte-dependent Autoantibody-induced Skin Blistering

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Leishmaniose cutânea na pele do dorso de Hamsters: um modelo útil para o rastreamento de drogas leishmanicidas

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Imunologia e Infecção

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imagem do HIV-1 Synapse Envelope induzida virológica e Sinalização em bicamadas lipídicas sintéticas

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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

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

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

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

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 ...

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 ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

Vasodilatação de isolado navios e o isolamento da matriz extracelular do Mice Tight-pele

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murino Transplante de pele de espessura total

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Desenvolvimento de um sistema de entrega de DNA Economical por &quot;Acufection&quot; e sua aplicação à Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Methods Article

Granulócitos-dependente Blistering pele induzida por auto-anticorpos