Name: lymphocyte isolation from human skin for phenotypic analysis and ex vivo cell culture
Uploaded: 2016-04-08T13:00:00
Description: Watch this Scientific Journal Video about Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture at JoVE.com

biopsias de piel de pacientes con psoriasis fueron amablemente proporcionados por el Dr. Andreas Koerber (departamento de Dermatología de la Universidad de Essen, Alemania) tras el consentimiento informado por escrito o por vía oral para uso científico. XH también es apoyado por SNCF 61263039 y 11101321 SNCF.

NOTA: Las biopsias de piel de individuos sanos se obtuvieron a partir de restos de piel abdominal de los individuos sometidos a cirugía plástica electiva tras el consentimiento informado por escrito o por vía oral para uso científico. El uso de piel humana fue aprobado y de acuerdo con las normas establecidas por los comités de ética médica para la investigación humana del centro médico de la Universidad Radboud, Nijmegen, Países Bajos y la Universidad de Essen, Alemania. 1. Preparac...

<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. 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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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A continuación, se presenta un protocolo para aislar eficazmente las células T residentes de la piel a partir de biopsias de piel humana. La ventaja de este protocolo es el aislamiento de un número relativamente alto de linfocitos viables, y la expresión de marcadores de superficie correspondientes. Los subconjuntos de células identificadas fueron:, células CD4 + y linfocitos T CD8 + y CD25 + Foxp3 + CD11c + DCS. Es importante destacar que la cultura ex vi...

La piel, como la interfaz principal entre el cuerpo y el medio ambiente, proporciona la primera línea de defensa contra la física externa, químicas y biológicas insultos tales como heridas, la radiación ultravioleta y los microorganismos. Piel comprende dos compartimientos principales, la epidermis y la dermis, y contiene una variedad de células inmunes incluyendo las células de Langerhans, macrófagos, células dendríticas (DC), y cerca de 20 millones de células T de memoria, casi dos veces el número presente en todo el volumen de sangre 1 , 2. Una creciente cantidad de datos apoyan la noción de que la piel tiene funciones inmunológicas esenciales, tanto durante la homeostasis del tejido y en diversas condiciones patológicas. Se cree que las células inmunes residentes en la piel normal para llevar a cabo la inmunovigilancia 3 y se ha demostrado que desempeñan un papel en el desarrollo de trastornos inflamatorios tales como psoriasis 4. En la piel lesional psoriásica, tanto CD4 + y CD8 + se infiltraron las células T eran observed y se demostró que la relación de la CD4 y CD8 varía en función del estado de la enfermedad 5. Sin embargo, estas poblaciones de células son difíciles de estudiar porque las técnicas actuales permiten el aislamiento de sólo unas pocas células. Las técnicas actualmente ampliamente utilizados para el aislamiento de células T de la piel humana se combinan disociación mecánica de la piel con tratamiento enzimático. Las biopsias de piel humana son ampliamente picada y se incubaron con enzimas como la tripsina, colagenasa y / o EDTA 6-8. Teniendo en cuenta que la piel es un tejido de barrera que es altamente resistente a las fuerzas de tracción y desagregación mecánica, los métodos establecidos de aislamiento de células T producen muy pocas células, e incluso un menor número de células viables, lo que hace ex vivo de cultivo de células de estas poblaciones de células difícil y desafiante. Aquí, se presenta un método modificado para aislar los linfocitos de la piel humana psoriásica saludable y participar mediante la combinación de mechandisociación ical de la piel usando un disociador de tejido automatizado en lugar del método establecido de picado ampliamente, junto con la digestión enzimática usando colagenasa. Se observaron varios subconjuntos de células inmunes viables, incluyendo países en desarrollo y las células T después de la preparación de una suspensión de una sola célula. Es importante destacar que la expresión de los marcadores de superficie CD3, CD4 y CD8 se conservó bien. Las células preparadas de este modo, están listos para su uso en cultivos de células ex vivo o análisis de citometría de flujo. Este protocolo se ha empleado con éxito para el análisis de biopsias de piel individuales (4 mm) derivadas de la piel lesional de pacientes con psoriasis. Los resultados mostraron que las células T de la piel del paciente residente producen citoquinas inflamatorias como la más IL-17 y IFN en comparación con voluntarios sanos 9.

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Name of Reagent/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Biopsy Punch</td>
			<td>Kai Europe</td>
			<td>BP-40F</td>
			<td>4 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable sterile scalpels</td>
			<td>Dalhausen</td>
			<td>1100000510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
			<td>Blue-capped, used as the dissociation tube.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">gentleMACS Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
			<td style="width:299px;">Automated tissue dissociator. Using the &#34;program spleen_01&#34; for dissociation of the skin biopsy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer&#160;</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">352350</td>
			<td>70 &#181;m nylon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96-well U-bottom plate</td>
			<td style="width:145px;">Greiner Bio-One</td>
			<td style="width:119px;">650180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>22409-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">GlutaMAX</td>
			<td>Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:281px;">Human Pooled Serum (HPS)</td>
			<td style="width:145px;">in house prepared</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Collagenase I</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C2674</td>
			<td>Type 1-A, suitable for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">DNase I</td>
			<td style="width:145px;">Calbiochem</td>
			<td style="width:119px;">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PBS</td>
			<td style="width:145px;">B Braun</td>
			<td style="width:119px;">3623140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">A4503-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fixable Viability Dye (FVD) APC-eFluo780</td>
			<td>eBioscience</td>
			<td>65-0865-18</td>
			<td style="width:299px;">Stain dead cells prior to cell fixation; dilute with PBS at 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Concentrate</td>
			<td>eBioscience</td>
			<td>00-5123-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation/Permeabilization Diluent</td>
			<td>eBioscience</td>
			<td>00-5223-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Buffer (10x)</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">BV421 Mouse anti-human CD45</td>
			<td style="width:145px;">BD</td>
			<td style="width:119px;">563879</td>
			<td>Clone: HI30; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">FITC Mouse anti-human CD14</td>
			<td>Dako</td>
			<td style="width:119px;">T0844</td>
			<td>Clone: TUK4; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PE Mouse anti-human CD56</td>
			<td style="width:145px;">Dako</td>
			<td style="width:119px;">R7127</td>
			<td>Clone: MOC-1; dilution factor 1:50</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:281px;">ECD Mouse anti-human CD3</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A07748</td>
			<td style="width:299px;">Clone: UCHT1; dilution factor 1:50 for surface staining; dilution factor 1:25 for intracellular staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PC5.5 Mouse anti-human CD4</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">B16491</td>
			<td>Clone: 13B8.2; dilution factor 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD11c</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A80249</td>
			<td>Clone: BU15; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC Mouse anti-human CD1c</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-903</td>
			<td>Clone: AD5-8E7; dilution factor 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">APC-AlexFluo700 Mouse anti-human CD8</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A66332</td>
			<td>Clone: B9.11; dilution factor 1:400</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:281px;">APC-AlexFluo750 Mouse anti-human CD19</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td style="width:119px;">A94681</td>
			<td>Clone: J3-119; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PeCy7 Mouse anti-human CD25</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">AD5-8E7</td>
			<td>Clone: BC96; dilution factor 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">&#160;PE Rat anti-human CLA</td>
			<td style="width:145px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-091-635</td>
			<td>clone: HECA-452; dilution factor 1:</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">eFluo450 Rat anti-human Foxp3</td>
			<td>eBIoscience</td>
			<td>48-4776-42</td>
			<td style="width:299px;">Clone: PCH101; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">AlexFluo488 Mouse anti-human IL-17A</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">53-7179-42</td>
			<td style="width:299px;">Clone: eBio64DEC17; intracellular staining; dilution factor 1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PeCy7 Mouse anti-human IFNg</td>
			<td style="width:145px;">eBioscience</td>
			<td style="width:119px;">25-7319-41</td>
			<td style="width:299px;">Clone: 4S.B3; intracellular staining; dilution factor 1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Flow-Count Fluorospheres</td>
			<td style="width:145px;">Beckman - Coulter</td>
			<td align="right" style="width:119px;">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

La piel humana tiene una función de barrera importante y contiene diversas células inmunes que contribuyen a la homeostasis del tejido y la protección contra los agentes patógenos. A medida que la piel es relativamente fácil acceso, que proporciona una plataforma ideal para estudiar los mecanismos de regulación inmune periférica. células residentes inmunes en sano conducta piel inmunovigilancia, pero también juegan un papel importante en el desarrollo de trastornos inflamatorios de la piel, tales como psoriasis. A pesar de ideas emergentes, nuestra comprensión de la biología subyacente de diversas enfermedades inflamatorias de la piel es aún limitada. Hay una necesidad de una buena calidad de las poblaciones (individual) de células aisladas a partir de muestras de piel biopsia. Hasta ahora, los procedimientos de aislamiento han sido seriamente obstaculizados por la falta de la obtención de un número suficiente de células viables. El aislamiento y el análisis posterior también se han visto afectados por la pérdida de marcadores de linaje de células inmunes, debido a la tensión mecánica y química causada por los procedimientos de disociación actuales para obtenersuspensión de células individuales. A continuación, describimos un método modificado para aislar las células T de la piel humana psoriásica saludable y participar mediante la combinación de disociación mecánica de la piel utilizando un disociador automatizado de tejidos y el tratamiento de colagenasa. Esta metodología conserva la expresión de la mayoría de los marcadores de linaje inmunes tales como CD4, CD8, Foxp3 y CD11c en la preparación de suspensiones de células individuales. Se muestran ejemplos de éxito de aislamiento de células T CD4 + y la posterior fenotípica y análisis funcional.

El protocolo que aquí se presenta producirá entre 2.200 ± 615 (media ± SEM, piel de voluntarios sanos) hasta 178.000 ± 760 (media ± SEM, piel lesionada de pacientes con psoriasis) linfocitos viables de la piel humana cuando se utiliza una sola biopsia de piel de 4 mm. Se identificaron diferentes tipos de células CD45 + en una sola célula suspensiones derivadas de la piel de individuos sanos, incluyendo las células T CD4 + (~ 45%), las 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.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The overall goal of this procedure is to efficiently isolate skin resident T cells from human skin biopsies. This is accomplished by first obtaining human skin biopsies using a round biopsy punch. Next, each skin biopsy is cut into four smaller pieces in a petri dish.Then, the skin tissue is mechanically and enzymatically dissociated to produce a single cell suspension. Finally, the skin resident lymphocytes are analyzed by flowing cytometry and cultured ex vivo. Ultimately, sufficient numbers of viable human skin resident lymphocytes can be isolated and analyzed by immunostaining and FACS analysis.This method will help to answer key questions in dermatology and skin tumor immunology. As for example, how resident T cells in the skin contribute to the disease. Demonstrating the procedure will be Xuehui He, a PhD student from our lab.After preparing culture medium, according to the text protocol, add two milliliters of complete medium to three wells of a sterile six well culture plate. Then, use a four millimeter round biopsy punch to obtain a skin biopsy. Place it in RPMI 1640 complete culture medium and leave them on the bench for up to four hours, or at four degrees Celsius over night.While the biopsy is incubating, label a blue capped dissociation tube and add five milliliters of complete culture medium. Next, use sterile tools to place the biopsy into the first well to rinse it. Transfer the tissue to the second well, rinse, and then repeat the process one more time in the third well.Transfer the well rinsed skin biopsy to a sterile petri dish and pipette 100 micro-liters of complete culture medium on top of the sample. Then, use a stainless steal disposable sterile scalpel to carefully scrape off the subcutaneous fat tissue. In a fresh sterile petri dish, cut each skin biopsy into four smaller pieces.Then transfer the samples to the prepared dissociation tube containing five milliliters of complete medium. Tightly cap the tube and attach upside down to the sleeve of the automated tissue dissociator. Make sure that all sample material is located in the area of the rotor.Next, start the dissociation process by running program M spleen zero one. To dissociate the biopsy at the appropriate rotating speed for 56 seconds. After processing, detach the dissociation tube from the dissociator and make sure that all the dissociated material is collected at the bottom of the tube.Add 150 micro-liters of collagenous one A to the dissociation tube and incubate the sample in a shaking water bath at 37 degrees Celsius for 60 minutes. Then, add 100 micro-liters of DNase into the dissociation tube and mix well. Attach the dissociation tube to the sleeve of the automated tissue dissociator and run program M spleen zero one to dissociate the biopsy one more time.Then, place a 70 micro-molar nylon cell strainer on the top of a 50 milliliter falcon tube and apply the dissociated sample materials to the cell strainer to remove cell clumps and tissue debris. Use five milliliters of complete medium to wash the cell strainer once. Then, centrifuge the filtrate at 450 G and 20 degrees Celsius, plus or minus two degrees for 10 minutes before aspirating the supernatant.After repeating the wash step one more time, use 300 micro-liters of complete culture medium to re-suspend the cell pallets. Single cell suspensions are now ready for further analysis. To carry out intracellular cytokine staining, use PMA, ionomysin, and brefeldin A to stimulate the cells and incubate at 37 degrees Celsius and five percent carbon dioxide for four hours before performing flow cytometry analysis.To perform polyclonal activation of skin resident T cells, aliquot 100 micro-liters of single cell suspension into a round bottom 96 well plate. Add anti-CD3, anti-CD28 mass antibody coated microbeads, recombinant human cytokine, IL-2 and IL-1B. With a well labeled weight lid, cover the culture plate and incubate at 37 degrees Celsius, five percent carbon dioxide, and 100%humidity.Then, when the color of the medium turns yellow, change it. A clear cell colony can be observed on day eight in cell culture. To carry out FACS analysis, transfer cells of interest into a V bottom 96 well plate.Centrifuge at 450 G and 20 degrees Celsius, plus or minus two degrees for two minutes. Then, aspirate the supernatant. Use 100 micro-liters of prepared fluorescent 780 conjugated fixable viability to dye to stain the cells by incubating at four degrees Celsius for 30 minutes.Then, add 100 micro-liters of FACS buffer and centrifuge before aspirating the supernatant. Next, after selecting the cell surface marker monoclonal antibodies from the list provided in the text protocol, use FACS buffer to prepare the antibody solutions. Use isotope control of the antibodies together with a non-stained sample to define gate settings.Now, add 25 micro-liters of prepared mouse antibodies mixture into each well and protect from light. Incubate at 20 degrees Celsius for 20 minutes. Add 100 micro-liters of FACS buffer, centrifuge, and aspirate the supernatant.Using buffers prepared for foxp3 staining according to the text protocol, add 100 micro-liters of fixation and permeabilization buffer to re-suspend the pallets. Mix well and incubate at four degrees Celsius for 30 minutes. Then, add 100 micro-liters of permeabilization buffer to each well and centrifuge before aspirating the supernatant.After washing the cells one more time and choosing the correct antibodies, add 20 micro-liters of prepared monoclonal antibody mixture into each well and protect from light before incubating at four degrees Celsius for 30 minutes. Using permeabilization buffer, wash the cells one more time. Then, use 110 micro-liters of FACS buffer to re-suspend the pallets and transfer the cell suspensions into mico FACS tubes.Finally, if the exact cell number is required, at 10 micro-liters of a well mixed uniform suspension of flurospheres to each sample immediately before taking FACS measurements. As shown here, different types of CD45 positive leukocytes were identified in single cell suspensions derived from skin of healthy individuals. Including CD4 positive T cells, CD8 positive T cells, and CD11c positive dendritic cells.Whereas few B cells, NK cells, or micro phages were observed. In addition, the methodology allows for the analysis of CD4 positive CD25 positive foxp3 positive cells in human skin biopsies. For example, by using intracellular staining after fixation and permeabilization, it is possible to demonstrate expression of the transcription factor foxp3 in CD25 positive T cells.As demonstrated here, to avoid background auto fluorescence and to avoid the auto fluorescent cell population, the anti-human CD45 mouse antibody conjugated to a brilliant violent 421 fluorochrome is recommended for gating of the lymphocyte population. This figure illustrates that the majority of resident cells in human skin were CD3 positive T cells. For cell viability analysis, a fixable viability dye is recommended to distinguish viable from dead or dying cells.For further functional analysis, by using a single four millimeter skin biopsy of lesional skin from psoriasis patients, it was shown that biopsy derived T cells produced IL-17a and inteferon gamma. Following a four hour stimulation with PMA ionomycin in the presence of brefeldin A.Finally, this figure shows that T cells from psyoriatic individuals have a much higher capacity to produce the psoriasis associated cytokines IL-17a and interferon gamma. Don't forget that working with human tissue and laboratory agents can be hazardous.Always take safety measures into account.

Research

JoVE Journal

Immunology and Infection

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

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

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

Aislamiento de linfocitos T humanos, cultura y análisis de la migración 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 ...

Investigación

Inmunología e Infección

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

HLA-Ig celdas en función de antígeno artificiales presentación para mejorar la eficiencia Ex vivo Ampliación de los Derechos Humanos CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

Preparación de suspensiones de una sola célula para el Análisis cytofluorimetric de diferentes regiones de piel de ratón

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

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Aislamiento y<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Las células γδ T, una célula αβT Progenitor Extrathymic

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Aislamiento y análisis de citometría de flujo de células inmunes del cerebro isquémico Ratón

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Murina de linfocitos Etiquetado por<sup&gt; 64</sup&gt; Cu-Anticuerpo objetivo de receptores de<em&gt; En Vivo</em&gt; Tráfico de células mediante PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Cuantificación de la Quimiotaxis de monocitos humanos In Vitro y linfocitos murinos, tráfico en Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Evaluación de la migración de linfocitos en un sistema de transmigración ex Vivo

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Aislamiento de células mononucleares tonitarias para estudiar las respuestas inmunitarias innatas ex vivo en un tejido linfático mucoso humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...