Name: an efficient and high yield method for isolation of mouse dendritic cell subsets
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Este trabalho foi financiado pelo NIH / NIAID AI45889 subvenção para SAP citometria de fluxo estudos foram realizados utilizando as instalações nucleares FACS apoiados pelo Cancer Center Einstein (NIH / NCI CA013330) e Center for AIDS Research (NIH / NIAID AI51519).

Experiências em animais são feitos de acordo com as directrizes aprovadas do comitê institucional cuidados com os animais e uso (IACUC). Todos os procedimentos que requerem a esterilidade são realizadas em uma cabine de segurança biológica. 1. Implantação de Melanoma B16.Flt3L em Ratos <ol><li> Cultura da linha expressando Flt-3 de células de melanoma B16 em frascos de cultura de tecidos T25, a uma densidade de 0,5 X 10 6 células / ml em meio DMEM completo com 10% de C...

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As células dendríticas são aceites como sendo a principal antigénio profissional células envolvidas na iniciação de respostas de células T apresentando. Sua função principal é fazer um levantamento do microambiente do tecido, tomando-se e processar antigénios para apresentação às células T. A fim de estudar a função dos subconjuntos de DC específicos, estes devem ser isolado em quantidade suficiente usando uma abordagem que mantém o seu fenótipo e funções normais. A maioria dos protocolos contar c...

As células dendríticas (DC) foram descobertos há quase quarenta anos como o &quot;grande estreladas celular (dendron grego)&quot; encontrado nos órgãos linfóides 1. Muitos estudos têm demonstrado que DC são as únicas células que apresentam o antigénio que pode estimular eficazmente células T ingénuas 2. Uma das principais funções destas células é a absorção de antigénios e apresentação e o seu processamento eficiente e carregamento destes para moléculas que apresentam antigénios. No baço de ratinho, as DCs pode ser separado em subconjuntos e plasmocitï¿½des convencionais. As DCs plasmocitoides são caracterizados por uma baixa expressão de CD11c e níveis elevados de B220 e Gr-1. Eles também são positivas para o mPDCA1 marcador de superfície e secretar interferon tipo I em resposta a toll like receptor 9 ligantes (TLR9). As DCs convencionais são elevados para CD11c e MHC classe expressão II. Eles podem ser divididos em três subgrupos distintos com base na expressão de superfície de marcadores fenotípicos tais como CD4, CD8a, DEC205, CD11b e receptor de células dendríticas 2 inibitória (DCIR2, reconhecida pelo anticorpo 33D1) proteínas de 3,4. Os CD8a Pos DCs são também conhecidos como CDC1, são positivos para DEC205, mas negativo para marcadores mielóides tais como CD11b e 33D1. O CD8a Neg DCs, também chamado cdc2, são positivos para 33D1, CD11b e CD4, mas falta DEC205. O subconjunto dupla negativa (isto é, negativo para CD4 e CD8a) é relativamente rara, e é negativo para DEC205 e 33D1. É o subconjunto menos caracterizado e pode ser uma forma menos diferenciadas de CD8a Neg DC. Diferenças fenotípicas nos diferentes subconjuntos de DC também se estendem para as suas funções in vivo. O CD8a Neg DCs são altamente fagocítica e são pensados ​​para apresentar antigénio exógeno principalmente por via do MHC de classe II às células T CD4 + 3. Em contraste, as DCs CD8a Pos são especializados para apresentação de antigénio proteico solúvel em MHC de classe Inum mecanismo denominado apresentação cruzada. O resultado da apresentação cruzada depende do estado de activação de estas DCs 5, e pode levar à expansão das células T citotóxicas (CTL) ou o desenvolvimento de células T reguladoras 6 2,7. Segmentação de antigénio para CD8a Pos DCs utilizando resultados de entrega anti-DEC205-anticorpo-mediadas, em grande parte na eliminação de células T 8, enquanto que a apresentação de antigénios derivados de células apoptóticas infectadas induz uma forte resposta de CTL 9. Além do reconhecimento de antigénios peptídicos, o sistema imunitário de mamífero evoluiu para reconhecer lípido e antigénios de glicolípidos. Estes antigénios são apresentados por moléculas de CD1, que são proteínas de MHC de classe I na superfície de células semelhantes que existem em várias formas relacionadas em vários mamíferos. Em ratos, um único tipo de molécula de CD1 CD1d altamente conservada chamada é responsável pela apresentação de antigénios glicolipidicos 10. O prefeito<html> população de células T que reconhecem os complexos glicolipidicos / CD1d é chamado células NKT invariantes (células iNKT). Estas células expressam um receptor de células T semi-invariante (TCR) composto por uma cadeia invariante TCRα que é emparelhado com cadeias TCRβ que a diversidade 11 limitadas. Ao contrário das células T convencionais que precisam proliferam e se diferenciam para se tornarem células T efectoras activadas, existem células iNKT como uma população efectora e começar a responder rapidamente após a administração de 12 glicolípido. Identificação de células apresentadoras de antigénio lípido fisiologicamente relevante é uma área activa de investigação, e vários tipos de células distintos, tais como células B, macrófagos e células dendríticas têm sido sugeridos para executar esta função. No entanto, demonstrou-se que o subconjunto CD8a Pos de DCS é a principal célula mediar a captação e a apresentação de antigénios a células lipídicas rato iNKT 13 e glicolípido mediada cross-priming de células T CD8 14. ove_content &quot;&gt; Para comparar a eficiência da apresentação de antigénio por diferentes células apresentadoras de antigénio, uma abordagem simples é a transferência de diferentes tipos de APC purificadas pulsadas com quantidades equivalentes de antigénio em hospedeiros sem tratamento prévio. experiências de transferência celular deste tipo são muitas vezes realizados para estudos imunológicos. no entanto, a realização de estudos de transferência com DCs tratadas ex vivo antigénio é desafiador, uma vez que estas células não existem populações como raros em órgãos linfóides, onde eles constituem menos do que 2% do total de células 15. por conseguinte, é necessário reforçar o desenvolvimento destas células em animais dadores para aumentar a eficácia dos protocolos de isolamento. Sabe-se que o linfóide comum e progenitores mielóides comuns, que são necessários para a geração de pdc e CD8 Pos subconjuntos e CD8 Neg DC, fms relacionados expressa a tirosina-cinase do receptor 3 (Flt-3). Após a in vivo, ligando Flt-3 (Flt-3L) administração, emigratião de Flt-3 que expressam as células progenitoras da medula óssea é aumentada, resultando no aumento da semeadura de órgãos linfóides periféricos e a expansão da sua descendência madura CC 16. A expressão de Flt-3 é perdida durante compromisso com o B, T ou NK vias de diferenciação celular. Portanto, apenas alterações mínimas são observados nestas células após administração Flt-3L. Expansão similar em populações DC é observada em ratinhos portadores de tumores gerados por implantação de uma linha celular de melanoma B16-secretoras de murino Flt-3L, que proporciona um método simples e económico para proporcionar níveis sistémicos sustentados de Flt-3L 17,18. Utilizando esta abordagem, temos desenvolvido um protocolo com base na implantação de células B16 de melanoma secretoras de Flt-3L para estimular a expansão de todos os subconjuntos normais DC em baço de ratinho, aumentando assim os rendimentos destas células que podem ser isolados por experiências subsequentes . Nós sempre achar que dentro de 10 - 14 dias após im subcutâneaplantação do tumor secretor de Flt-3, os murganhos desenvolvem esplenomegalia com enriquecimento acentuada das DCs para constituir 40 - 60% do total de células de baço. A partir destes baços, diferentes subconjuntos de DC pode ser isolado com pureza elevada utilizando kits de purificação de células padrão disponíveis comercialmente que utilizam marcadores fenotípicos específicos do subconjunto.

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			<td>0.05% Trypsin-EDTA&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>25300-054</td>
			<td></td>
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			<td>Isoflurane&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>CDS019936-250MG</td>
			<td></td>
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			<td>Collagenase D&#160;</td>
			<td>Roche Diagnostics</td>
			<td>11088858001</td>
			<td></td>
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		<tr>
			<td>DNase I (dry powder)</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
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		<tr>
			<td>200 proof ethanol&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>9-6705-004-220</td>
			<td>Used to prepare 70% ethanol</td>
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			<td>RBC lysis buffer</td>
			<td>Sigma-Aldrich</td>
			<td>R7757</td>
			<td></td>
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			<td>RPMI-1640 medium with L-glutamine&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11875-119</td>
			<td></td>
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			<td>DMEM medium with L-glutamine&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11995-073</td>
			<td></td>
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			<td>200 mM L-glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td></td>
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			<td>MEM non-essential amino acids&#160;</td>
			<td>Life Technologies, Gibco</td>
			<td>11140-050</td>
			<td></td>
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			<td>MEM essential amino acids&#160;</td>
			<td>Life Technologies</td>
			<td>11130-051&#160;</td>
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			<td>&#946;-mercaptoethanol&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>21985-023</td>
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			<td>Sodium pyruvate&#160;</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
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			<td>HEPES&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15630</td>
			<td></td>
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			<td>Phosphate buffered saline (PBS Ca2+ and Mg2+ free pH 7.2)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>20012-050</td>
			<td></td>
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			<td>Dulbecco&#8217;s PBS (DPBS with Ca2+ and Mg2+)</td>
			<td>Life Technologies, Gibco</td>
			<td>14040-182</td>
			<td></td>
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			<td>0.5 M Ethylenediaminetetraacetate (EDTA) solution&#160;</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
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			<td>Bovine serum albumin (BSA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
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			<td>Fetal calf serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11050&#160;</td>
			<td></td>
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			<td>Penicillin/streptomycin&#160;</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15140-163</td>
			<td></td>
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			<td>Trypan blue (dry powder)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6146-5G&#160;</td>
			<td>Prepare 0.08% with PBS</td>
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			<td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td>
			<td>Miltenyi Biotech</td>
			<td>130-092-786</td>
			<td></td>
		</tr>
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			<td>Magnetic beads conjugated with anti-mouse CD11c&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-152-001</td>
			<td></td>
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			<td>CD8&#945;Pos mouse DC isolation kit&#160;</td>
			<td>Miltenyi Biotech</td>
			<td>130-091-169</td>
			<td></td>
		</tr>
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			<td>Fc-gamma receptor blocking antibody (Clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c-FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>553801</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945;-PerCP&#160;</td>
			<td>BD Biosciences</td>
			<td>553036</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse B220-PE&#160;</td>
			<td>BD Biosciences</td>
			<td>553090</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes&#160;</td>
			<td>BD</td>
			<td>26048</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G1 needle&#160;</td>
			<td>BD</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dishes&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments&#160;</td>
			<td>Kent Scientific</td>
			<td>INSMOUSEKIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m&#160;</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Petri plates&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filtration system (500 ml(0.22 um&#160;</td>
			<td>Corning</td>
			<td>431097</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns&#160;</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand MACS separator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-bore 200 &#956;l pipette tips&#160;</td>
			<td>PerkinElmer</td>
			<td>111623</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning ultra-low attachment 96-well plates&#160;</td>
			<td>Corning</td>
			<td>CLS3474-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>6-8 week old female C57BL/6 mice&#160;</td>
			<td>Jackson Research Laboratories, Jax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murine B16.Flt3L melanoma cell line</td>
			<td></td>
			<td></td>
			<td>described by Mach et al. ,2000</td>
		</tr>
		<tr>
			<td>Serum free DMEM and RPMI media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine 
			 
			5 ml MEM nonessential amino acids (100x, 10 mM)&#160;&#160;&#160;&#160; 
			 
			5 ml HEPES Buffer (1 M)&#160;&#160;&#160;&#160; 
			 
			5 ml L-glutamine (200 mM)&#160; 
			 
			0.5 ml 2-mercaptoethanol (5.5 x 10-2 M)</td>
			<td></td>
			<td></td>
			<td>Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need.&#160; Filter sterilize the media by passing through a 0.22 &#181;m vacuum filtration system.</td>
		</tr>
		<tr>
			<td>Complete RPMI and DMEM media</td>
			<td></td>
			<td></td>
			<td>Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.</td>
		</tr>
		<tr>
			<td>MACS buffer:&#160;</td>
			<td></td>
			<td></td>
			<td>Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca2+ and Mg2+ Free, pH 7.2) and 2 ug/ml 2.4G2 (Fc-Block antibody).&#160;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer</td>
		</tr>
		<tr>
			<td>10x Collagenase D and DNase 1 stock solution 
			Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca++ and Mg++ to obtain a solution of approximately 1,000-</td>
			<td></td>
			<td></td>
			<td>2,000 Units of collagenase activity per ml 
			 
			This 10x stock solution can be prepared ahead of time and stored at -20 &#176;C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use</td>
		</tr>
	</tbody>
</table>

an efficient and high yield method for isolation of mouse dendritic cell subsets

As células dendríticas (CDs) são células apresentadoras de antigénios profissionais primariamente responsável pela aquisição, processamento e apresentação de antigénios no antigénio que apresenta moléculas para iniciar a imunidade mediada por células-T. As células dendríticas podem ser separados em vários subconjuntos fenotipicamente e funcionalmente heterogéneas. Três subconjuntos importantes de células dendríticas esplênicas são plasmocitï¿½des, as células CD8a POS e CD8a Neg. As DCs plasmocitoides são produtores naturais de interferão de tipo I e são importantes para a imunidade de células T anti-viral. O subconjunto de CD8a Neg DC é especializado para apresentação de antígenos MHC classe II e está envolvido no centro de priming células T CD4. O CD8a Pos DCs são os principais responsáveis ​​para a apresentação cruzada de antígenos exógenos e priming células T CD8. As DCs CD8a Pos ter sido demonstrado ser mais eficaz na apresentação de antigénios de glicolípidos por moléculas CD1d a um cel t especializadol população conhecidas como células invariante natural killer T (iNKT). A administração de ligando Flt-3 aumenta a frequência de migração de células progenitoras de células dendríticas a partir da medula óssea, resultando em última análise, a expansão de células dendriticas em órgãos linfóides periféricos em modelos murinos. Temos este modelo adaptado para purificar grandes números de células dendríticas funcionais para utilização em experiências de transferência de células para comparar proficiência in vivo de diferentes subconjuntos de DC.

O resultado deste procedimento baseia-se na ampliação de subconjuntos de DC de murino por Flt-3L expressos pelas células de melanoma implantado. O tumor B16.Flt3L foi derivado a partir de um ratinho C57BL 6 /, e deve ser implantado num animal com essa estirpe de fundo, a fim de evitar o fracasso do tumor para se estabelecer devido à rejeição. Em alguns casos, pode ser desejável a utilização de ratinhos geneticamente modificados para derivar as DCs com defeitos conhecidos nas via...

Watch this Scientific Journal Video about An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets at JoVE.com

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

The overall goal of this protocol, is to develop of a high efficiency, high yield method for generating dendritic cell or DCs, that fatefully reflect their in-vivo phenotypic and functional divergence. Dendritic cells exist as great populations in lymphoid organs. Therefore, we use filter ligand to increase the frequency of these essential cells in donor tissues to facilitate that isolation.The main advantage of this technique is that it removes generation of all the DC subsets in suitable numbers for analysis using only the commercially available agents. To harvest flt-3 ligand expressing B16 melanoma cells from a 90%confluent culture, first wash the cells with PBS and incubate the culture with 05%trypsin at 37 degrees Celsius. After 5 minutes, quench the protease activity with 20 milliliters of 4 degrees Celsius complete dima medium and detach the adherent cell model layer with light tapping and gentle pipetting.Collect the cells by centrifugation and count them. Then, re-suspend the single cell suspension to a 1 times to the 8 cells per milliliter concentration in PBS for their subcutaneous injection into C57 black 6 recipient mice. When the tumor reaches two to ten milliliters in diameter, harvest the spleens from the tumor bearing animals and place the spleens in a petri dish containing fresh RPMI medium.Using a scalpel, cut the tissues into 0.2 square centimeters pieces, and then incubate the fragments in ten milliliters of collagenase and Dnase at 37 degrees Celsius. After 30 minutes, pour the partially digested tissue slurry onto a 70 micron cell strainer, and use a five milliliter syringe plunger to press the remaining tissue fragments through the mesh of the strainer. Rinse the filter with 10 milliliters of fresh medium, pooling the wash with the rest of the single cell suspension and pellet the cells.We suspend the cell pellet in 2 milliliters of red blood cell lysis buffer at room temperature for 10 minutes. Then wash the cells in 10 milliliters of fresh RPMI medium, and re-suspend them at 1 times 10 to the 8 cells per milliliter in cell sorting buffer containing FC block. To isolate the plasma cytode DCs, thoroughly mix 300 microliters of biotin-labelled antibody cocktail from a plasma cytode DC isolation kit with 200 microliters of the splenic single cell suspension.Place the cells in an ice water bath for 15 minutes, with gentle tapping every three minutes. Then wash the cells with 12 milliliters of cell sorting buffer, and re-suspend the pellet in 500 microliters of fresh sorting buffer. Next, incubate the cells in 300 microliters of anti-biotin, antibody conjugated beads for another 15 minutes in the ice water with regular tapping.After washing the cells in fresh sorting buffer, re-suspend the pallet in 2 milliliters of cell sorting buffer, and load an appropriately sized magnetic column onto its corresponding magnet. Equilibrate the column with five milliliters of fresh 4 degree Celsius cell sorting buffer. When all of the buffer has drained from the top of the column, carefully add the cells to the center of the surface of the column head, and collect the flow-through.Then wash the column with 10 milliliters of fresh, 4 degrees Celsius sorting buffer and collect the flow-through in the same tube. After passage though a second magnetic column, a plasma cytode dendritic cell population with a greater than 94%purity can be expected. To isolate the CD8aplha+and CD8alpha-DCs, mix the rest of the splenic cell suspension with 100 microliters of biotion conjugated antibody cocktail from a CD8alpha+dendritic cell isolation kit for 15 minutes on ice with gentle tapping, as just demonstrated.At the end of the incubation, wash the cells in 12 milliliters of 4 degrees Celsius cell sorting buffer, and re-suspend the pellet in 150 microliters of fresh 4 degrees Celsius cell sorting buffer. Next, mix the cells with 100 microliters of the CD8alpha+dendritic cell isolation kit anti-biotin beads. After 15 minutes on ice with gentle tapping, wash the cells in 10 milliliters of 4 degrees Celsius cell sorting buffer, and re-suspend the pellet in 1 milliliter of fresh 4 degrees Celsius sorting buffer.At the end of the incubation, sort the cells by magnetic bead separation, as just demonstrated, collecting the flow-through and the first wash. Then, spin down the cells and re-suspend the pellet in one milliliter of fresh 4 degrees Celsius cell sorting buffer. Now label the cells with 200 microliters of anti-CD8aplha conjugated magnetic beads from the kit, and pass the cells through a new column, collecting the CD8alpha-dendritic cell flow-through.To collect the CD8alpha+DCs, transfer the column into a 15 milliliter conical tube, and plunge 5 milliliters of fresh 4 degrees Celsius cell sorting buffer though the column. After the running the cells through a second column, a greater than 96%CD8alpha+dendritic cell population can be expected. To isolate the CD8alpha-cells, spin the the CD8alpha-flow-through cell suspension.Then label the cells with 100 microliters of anti-CD11 c-magnetic beads and collect the magnetic bead positive cell population, as just demonstrated, to obtain a greater than 97%CD8alpha-dendritic cell subset population. Finally, determine the number of live cells, by trypan blue exclusion. Once the tumor becomes palpable, the B16 flt-3 ligand tumor bearing mice consistently exhibit a dramatic increase in the splenic cellularity that correlates with the expansion of the splenic dendritic cell subsets.Interestingly, while a 15 to 20-fold increase in the absolute cell numbers is observed for the conventional DCs in these animals, only about a 4-fold increase is observed for the plasma cytode DC subset. The different dendritic cell subsets are characterized according to their B220, CD11b, CD11c and CD8alpha expression. The cell populations also exhibit other unique subset markers, however, with mPDCA1 expressed exclusively on the plasma cytode DCs DEC205 highly expressed on the CD8alpa+DCs, and CD11b and 33D1 detected most prominently on the CD8alpha-DCs.Using this method, we have demonstrated CD8alpa+DCs to be the most efficient in the presentation of antigens by CD bonding molecules to a specialized T-cell population known as invariant NKT cells. The most critical feature of DC isolation is to maintain strict sterility and antidoxant-free conditions. During the procedures, these T-cells are highly responsive to antidoxant.While attempting this procedure, it is important to remember to handle the cells gently, as repeated mechnanical stimulation may alter the maturation state of dendtritic cells. Once mastered, this technique can be used to isolate a high number of all the major DC subsets from single spleen. Thanks for watching and good luck with your experiments.

Research

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