This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG14593, MFAG13235), the Fondazione Cariplo (Grant 2010-0678 and NANOVAC) and the Fondazione Regionale per la Ricerca Biomedica.

연구 승인 : 실험 프로토콜은 Decreto에​​ 따라 건강의 이탈리아 교육부 (로마, 이탈리아)의 승인을 하였다는 N, 27 gennaio 1992 legislativo. (116) &quot;Attuazione 델라 Direttiva N. 609분의 86 / CEE의 본초 디 protezione 글리 animali utilizzati 피니 sperimentali 오 광고 altri 피니 scientifici.&quot; 1. 피부 샘플을 얻기 <ol><li> 자궁 경부 전위로 마우스를 안락사. </li><li> 귀: <ol><li> 마우스의 귀 털이없는 부분을 잘라. </li><li> 집게로 떨어져 당겨 분리 지느러미와 복부 측면. 부드럽게 집게로 귀 안쪽 부분을 긁어로 남아있는 연골을 제거합니다. </li></ol></li><li> 트렁크 피부 : <ol><li> 전기 면도기와 섬세한 제모 로션 두 동물의 전체 피부를 면도. </li><li> (2cm 2의 전체 동물의 피부까지) 원하는 피부 샘플을 잘라. <ol><li> 전체 지느러미를 절단하는 경우와 VENTRAL 마우스 피부는 다시 다음 마우스 본체 주변의 면도 영역의 국경을 따라 절단 한 마우스의 중앙에 수직 절단을합니다. </li><li> 조심스럽게 수술 가위 나 메스의 폐쇄 라운드 예리하게 팁을 피부 샘플을 분리하면서 집게로 여전히 피부 샘플을 유지, 복막 다시 근육으로부터 피부를 분리합니다. </li></ol></li><li> 빙냉 PBS 10 ㎖를 함유하는 100mm 세포 배양 플레이트를 준비한다. </li><li> 단계 1.3.3에서 제조 한 접시에 PBS의 피부 샘플을 침수와 집게로 피부 샘플을 긁어하여 피하 지방을 제거합니다. </li></ol></li><li> 꼬리: <ol><li> 마우스의 꼬리를 잘라. </li><li> 수술 용 가위 외과 메스, 꼬리베이스에서 시작하여 꼬리의 끝 부분에 도달와 꼬리에 수직 절단합니다. </li><li> 꼬리으로부터 피부를 분리 집게를 사용합니다. 꼬리의 기지에서 절단의 가장자리를 잡고하고 채 끝을 향해 조심스럽게 잡아 당겨집게와 꼬리의 몸. </li><li> 부드럽게 피부 샘플을 긁어에 의해 어떤 여분의 지방을 제거합니다. </li></ol></li><li> 노상 강도 <ol><li> 수술 가위로 전체 마우스 발을 잘라. 모든 털이 피부를 절단 방지. </li><li> 숫자의 시작 발목에서 길이 방향 다리의 피부를 잘라. </li><li> 집게로 피부를 잡는 동안 집게 또는 손으로 발목 뼈를 잡고 장갑을 벗고 것처럼 자리를 향해 피부를 잡아 당깁니다. </li></ol></li></ol> 2. 소화 <ol><li> 효소 믹스 (사양 재료 목록 참조) 300 μg의 / ㎖, DNAse1 50 U / ㎖ 다음 레시피 (재료 목록에서와 같이 제조) 원액에서 소화 칵테일을 준비합니다. RPMI 5 % FBS에 희석. </li><li> (트렁크 피부 : 피부의이 소화 칵테일 ml의 모든 2cm 2, 최대 3 ㎖ 총) 소화 칵테일 2 ㎖와 35mm 페트리 접시에 작은 조각으로 가위로 피부 샘플을 잘라. </li><li>90 분 동안 37 ° C에서 품어. 조심스럽게 배양시 배양 접시를 1-2 번 흔들어. </li><li> RPMI1640 5 % FBS 1 ㎖를 함유하는 66mm 페트리 접시에서 70 μm의 셀 스트레이너에 소화 된 피부 샘플을 붓는다. </li><li> RPMI 1640 5 % FBS의 5 ㎖로 스트레이너 씻으십시오. </li><li> 주사기 플런저 셀 스트레이너를 통해 샘플을 짠다. </li><li> 세척 플런저, 페트리 접시와 RPMI1640 5 % FBS 20 ㎖와 스트레이너, 50 ㎖ 튜브에 전송할 수 있습니다. </li></ol> 3. 항체 및 라벨링 <ol><li> PBS 1 % FBS (또는 BSA)을 5 ml로 두 번 얻어진 현탁액을 씻으십시오. </li><li> 원하는 항체 얻어진 세포 현탁액 레이블. <ol><li> 100 μL에 재현 탁 샘플 / PBS 1 % FBS에 희석 aCD16 / CD32 (클론 2.4G2) 2 ㎍ / ml를 함유하는 혼합물의 샘플. </li><li> Fc 수용체를 통해 원하는 항체의 비특이적 결합을 차단하기 위해 4 ℃에서 15 분 동안 인큐베이션. </li><li> 표에 표시된 항체를 추가1. <ol><li> 4 μg / ml의 농도 PBS, 1 % FBS 100 ㎕ / 샘플의 원하는 항체를 희석 혼합하여 준비한다. </li><li> 튜브에서 2 μg / ML의 최종 농도로 항체 혼합물의 200 μL의 부피가되도록 각 시료에 3.2.3.1 단계에서 제조 된 혼합물 100 ㎕를 추가한다. </li></ol></li><li> 4 ° C에서 30 분 동안 품어 빛에서 다룹니다. </li></ol></li><li> 400 XG에서 5 분 동안 PBS 1 % FBS 1 ㎖, 원심 분리로 세척 PBS 1 % FBS 300 μL의 상층 액과에 resuspend 폐기합니다. </li><li> 2 μg / ML의 최종 농도에서 DAPI를 추가하고 10 분 동안 빛으로부터 보호, 4 ° C에서 배양한다. </li><li> FACS에 의해 얻어진 샘플을 분석 할 수 있습니다. <ol><li> 백혈구를 분리하기 위해, 다음 게이팅 전략을 사용 <ol><li> 먼저 DAPI- 인구에 게이팅에 의해 죽은 세포를 제거한다. </li><li> 그림 1에 나타낸 바와 같이, FSC의 W 플롯 대 FSC-A에서 선택 단봉 </strong&gt;. </li><li> 게이트 주목 백혈구의 전형적인 크기와 입도에 기초하여 상기 셀. </li><li> 그림 1에 나타낸 바와 같이 FSC-A CD45 플롯에서 CD45 + 세포를 선택합니다. </li></ol></li><li> 다음과 같이 표면 마커의 발현에 의해 백혈구의 다양한 인구를 구별 : <ol><li> 중 CD11c + MHC II + 세포로 수지상 세포를 식별합니다. 이들은 나중에 CD207- 피부 수지상에서 랑게르한스 세포 및 피부 CD207 + 수지상 세포를 구별하는 CD207의 발현에 대해 분석 할 수있다. </li><li> CD64 + F4 / 80 + 세포로서 대 식세포를 식별합니다. </li><li> CD19 + MHCII + 세포와 같은 B 세포를 식별합니다. </li><li> CD8 + 또는 CD4 + 세포와 같은 T 세포를 식별합니다. </li></ol></li></ol></li></ol>

<ol>
	<li>Streilein, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuits+and+signals+of+the+skin-associated+lymphoid+tissues+(SALT).">Circuits and signals of the skin-associated lymphoid tissues (SALT).</a> Journal of Investigative Dermatology. 85 (1 Suppl), 10s-13s (1985).</li><li>Tay, S. S., Roediger, B., Tong, P. L., Tikoo, S., Weninger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Skin-Resident+Immune+Network.">The Skin-Resident Immune Network.</a> Current dermatology reports. 3, 13-22 (2014).</li><li>Shen, W., 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=Adaptive+immunity+to+murine+skin+commensals.">Adaptive immunity to murine skin commensals.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (29), E2977-E2986 (2014).</li><li>Broggi, A., Zanoni, I., Granucci, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migratory+conventional+dendritic+cells+in+the+induction+of+peripheral+T+cell+tolerance.">Migratory conventional dendritic cells in the induction of peripheral T cell tolerance.</a> Journal of leukocyte biology. 94 (5), 903-911 (2013).</li><li>Pan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+metabolize+sunlight-induced+vitamin+D3+to'program'T+cell+attraction+to+the+epidermal+chemokine+CCL27.">DCs metabolize sunlight-induced vitamin D3 to'program'T cell attraction to the epidermal chemokine CCL27.</a> Nature Immunology. 8 (3), 285-293 (2007).</li><li>Vitali, C., 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=Migratory,+and+not+lymphoid-resident,+dendritic+cells+maintain+peripheral+self-tolerance+and+prevent+autoimmunity+via+induction+of+iTreg+cells.">Migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of iTreg cells.</a> Blood. 120 (6), 1237-1245 (2012).</li><li>Azukizawa, H., 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=Steady+state+migratory+RelB++langerin++dermal+dendritic+cells+mediate+peripheral+induction+of+antigen-specific+CD4++CD25++Foxp3++regulatory+T+cells.">Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells.</a> European journal of immunology. 41 (5), 1420-1434 (2011).</li><li>Idoyaga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+role+of+migratory+dendritic+cells+in+peripheral+tolerance+induction.">Specialized role of migratory dendritic cells in peripheral tolerance induction.</a> The Journal of clinical investigation. 123 (2), 844-854 (2013).</li><li>Boyman, O., Conrad, C., Tonel, G., Gilliet, M., Nestle, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+role+of+tissue-resident+immune+cells+in+psoriasis.">The pathogenic role of tissue-resident immune cells in psoriasis.</a> Trends in immunology. 28 (2), 51-57 (2007).</li><li>Di Meglio, P., Perera, G. K., Nestle, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multitasking+organ:+recent+insights+into+skin+immune+function.">The multitasking organ: recent insights into skin immune function.</a> Immunity. 35 (6), 857-869 (2011).</li><li>Rosenblum, M. D., 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=Response+to+self+antigen+imprints+regulatory+memory+in+tissues.">Response to self antigen imprints regulatory memory in tissues.</a> Nature. 480 (7378), 538-542 (2011).</li><li>Rosenblum, M. D., Truong, H. A., Abbas, A. K., Gratz, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=177:+Maintenance+of+memory+regulatory+T+cells+in+peripheral+tissues.">177: Maintenance of memory regulatory T cells in peripheral tissues.</a> Cytokine. 63 (3), 284-285 (2013).</li><li>Kim, B. S., 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=TSLP+elicits+IL-33-independent+innate+lymphoid+cell+responses+to+promote+skin+inflammation.">TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation.</a> Science translational medicine. 5 (170), (2013).</li><li>Gopinathan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+skin+structure:+scratching+the+surface.">New insights into skin structure: scratching the surface.</a> Advanced drug delivery reviews. 54 (Suppl 1), S3-S17 (2002).</li><li>Sandrine, H., 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=CD207++CD103++dermal+dendritic+cells+cross-present+keratinocyte-derived+antigens+irrespective+of+the+presence+of+Langerhans+cells.">CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.</a> The Journal of Experimental Medicine. 207 (1), 189-206 (2010).</li><li>Bernard, M., Samira, T., Sandrine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+functions+of+dendritic+cells+and+macrophages+in+the+skin.">The origins and functions of dendritic cells and macrophages in the skin.</a> Nature Reviews Immunology. 14 (6), 417-428 (2014).</li></ol>

The authors declare that they have no competing financial interests.

우리는 다른 마우스 피부 영역에서 단일 세포 현탁액을 제조하는 방법을 설명 하였다. 우리가 채택 소화 방법은 높은 수율을 제공하지만, 또한 다음 FACS 분석을 위해 근본적인 표면 마커의 발현을 유지 아닙니다. 콜라게나 제 I 및 II와 써모의 칵테일의 사용은,이 방법은 재현성이 높은 만드는 효소 활성의 배치의 차이에 최소 배치를 보장합니다. 다른 공개 방법은 다른 효소 ?...

The skin is one of the largest organs of the body and its large surface is continuously exposed to the external environment. Therefore the skin has to protect the organism from potential threats in order to maintain homeostasis, both by physical means, and by providing active protection toward potential pathogen entry. In a similar way to the gut and lung mucosa, the skin homes a variety of immune cells that continuously interact with the epithelium in order to maintain immune surveillance. This complex system that involves both immune and non-immune cells of the skin has been acknowledged since the early days of immunology, when in 1978 the term "Skin-associated lymphoid tissue" (SALT) 1 was first coined to describe such complexity. 
The large number of immune cells residing in the skin helps the organism not only to fight potential invasions, but also to orchestrate wound healing and to maintain tolerance toward self-antigens and the skin microbiota 2,3. 
Amongst skin resident immune cells, Dendritic Cells (DCs) have a crucial role in shaping immune responses and maintaining homeostasis 4. Dermal DCs and Langerhans cells readily respond to pathogen invasions and, upon migration to lymph nodes activate T cells and induce the expression of skin homing receptors on the newly generated effector T cells 5. DCs also have a fundamental role in regulating skin homeostasis. DCs migrating from the dermis to the lymph nodes in homeostatic conditions transport skin sequestered antigens with the purpose of inducing T cell tolerance mostly through the differentiation of regulatory T cells (Tregs) specific for skin antigens 6-8. 
T cells represent the most abundant population of immune cells in the skin. Not only do they infiltrate the skin during an infection but they also represent a stable population among skin resident lymphocytes 9,10. Both CD8+ and CD4+ resident memory (rm)T cells reside in the skin and, following an infection, can respond long before effector T cells are recruited from the blood. In the skin, also resides a population of tissue resident Tregs capable of maintaining tolerance toward skin antigens and tissue homeostasis. These cells are rapidly and potently activated after exposure to their cognate antigen and have therefore been defined as memory Tregs 11,12. 
Along with DCs and T cells, many innate immune cells, such as NK cells, gamma delta T cells, group 2 ILCs 13, Mast Cells and Macrophages, populate the skin and contribute to host protection. To analyze such a complex environment, it is mandatory to obtain single-cell suspensions from skin specimens with high efficiency while at the same time preserving the expression of surface markers for flow cytofluorimetric analysis or sorting. 
Murine skin presents an outer epidermal layer, constituted mainly of keratinocytes, Langerhans cells and dendritic epidermal T cells; and the dermal layer beneath. The dermis homes most of the immune cells and is made by an extracellular matrix in which collagen fibers are the most abundant, especially collagen-I and collagen-IV. Unlike human skin, mouse skin is covered in fur, and thus more populated with hair follicles. It is, also, much thinner than human skin and contains a muscular layer, the panniculus carnosus, which helps wound healing 14. These characteristics render it more difficult to efficiently disrupt the collagen net of the dermis in order to get immune cells out.
In this paper we provide a suitable method for digesting the extracellular matrix that relies on a cocktail of high purity collagenase 1 and 2 and thermolysin. Moreover, since the analysis of skin from different regions requires different experimental procedures, we will show different methods to efficiently obtain skin samples from the footpad, tail, ear and trunk. We will then label the samples in order to evaluate the presence of DCs (MHCII+CD11c+CD45+) Macrophages (CD11b+F4/80+CD45+), CD8+, and CD4+ T cells.

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:203px;">Reagents</td>
			<td style="width:88px;"></td>
			<td style="width:155px;"></td>
			<td style="width:276px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:203px;">Supplemented RPMI 1640 medium&#160;</td>
			<td></td>
			<td></td>
			<td style="width:276px;">RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">FBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Liberase TM (Enzyme Mix)</td>
			<td>Roche</td>
			<td>5401119001</td>
			<td style="width:276px;">resuspend [2.5mg/ml] in dH20 store aliquots at -20&#176;C&#160;</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Dnase I</td>
			<td>Sigma&#160;</td>
			<td>D4263-1VL</td>
			<td style="width:276px;">resuspend in RPMI 1640 w/o serum [1000u/ml]</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Surgical Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">35mm petri dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">66mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">75um Cell Strainers</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company</td>
			<td>Clone</td>
			<td>Label</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD45.2</td>
			<td></td>
			<td>104</td>
			<td>PE-Cy5.5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD11c</td>
			<td></td>
			<td>N418</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CD11b</td>
			<td></td>
			<td>M1/70</td>
			<td>FITC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">F4/80</td>
			<td></td>
			<td>A 3-1</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD4</td>
			<td></td>
			<td>Gk1.5</td>
			<td>APC-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD8</td>
			<td></td>
			<td>53-6.7</td>
			<td>PE</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD64</td>
			<td></td>
			<td>X54-5/7.1</td>
			<td>PE-Cy7</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">aCD207</td>
			<td></td>
			<td>4C7</td>
			<td>APC</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Digestion cocktail</td>
			<td></td>
			<td></td>
			<td>Dilute in RPMI 5%FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LIberase TM&#160;</td>
			<td></td>
			<td></td>
			<td>300 ug/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DNAse1&#160;</td>
			<td></td>
			<td style="width:155px;"></td>
			<td>50 U/ml</td>
		</tr>
	</tbody>
</table>

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.

신체의 다른 부위에서 피부 표시된 항체와 설명 및 스테인드 프로토콜에 따라 소화했다. 게이팅 전략은 먼저 단봉 (FSC-W 대 FSC-A)와 림프구의 형태 (SSC-A 대 FSC-A)와 세포에서 방송 한 다음 세포 (DAPI- 세포), 게이트를 선택 그림 1에 도시되어있다. 표시 할 때, 조혈 원점에서 CD45 + 세포가 선택됩니다. DC가이 항...

Watch this Scientific Journal Video about Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions at JoVE.com

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

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.

다른 마우스 피부 지역에서 Cytofluorimetric 분석을위한 단일 세포 현탁액의 준비

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

preparation-single-cell-suspensions-for-cytofluorimetric-analysis

Research

JoVE Journal

Immunology and Infection

18.1K 조회수.  University of Milano-Bicocca. 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. 

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

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

실험 면역 Hypophysitis의 유도를위한 마우스 뇌하수체 Immunogen의 준비

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

연구

면역학 및 감염병학

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids에서 바이러스성 DNA의 준비

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Granulocyte-dependent Autoantibody-induced Skin Blistering

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA 1. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies 2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA 1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.

과립에 따라 달라 Autoantibody 유발 피부 젖어

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

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T 세포 양적 높은 처리량 단일 세포 세포 독성 분석

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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.

표현형 분석 및 대한 인간의 피부에서 림프구 분리<em&gt; 전의 VIVO</em&gt; 세포 배양

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

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.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

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

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

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

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4의 준비<sup&gt; +</sup&gt; 현미경으로 GD3 및 GD2 강글 리오 시드 멤브레인 식의 분석을위한 T 세포

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Immunophenotype 및 Cytokine 생산 대량 Cytometry 통해 주변 전 혈의 단일 세포 분석

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...