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.

אישור לימוד: פרוטוקולי הניסוי אושרו על ידי משרד הבריאות האיטלקי (רומא, איטליה) על פי Decreto legislativo 27 gennaio 1992, n. 116 &quot;Attuazione דלה Direttiva n. 86/609 / CEE ב מאטריה di protezione degli animali utilizzati פיני sperimentali o המודעה אלטרי פיני scientifici.&quot; 1. קבלת דגימות עור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר על ידי נקע בצוואר הרחם. </li><li style=";text-align:right;direction:rtl"> אֹזֶן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את החלק ללא שיער של האוזניים של העכבר. </li><li style=";text-align:right;direction:rtl"> הגב נפרד בצד הגחון על ידי משיכת אותם לגזרים עם מלקחיים. הסר את כל סחוסים הנותרים על ידי גירוד החלק הפנימי בעדינות של אוזניים עם מלקחיים. </li></ol></li><li style=";text-align:right;direction:rtl"> עור Trunk: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגלח את העור כולו של החיה שניהם עם סכין גילוח חשמלי ועם קרם מקריח עדין. </li><li style=";text-align:right;direction:rtl"> חותך את מדגם העור הרצוי (מ -2 סנטימטרים 2 עד עורות בעלי החיים השלמים). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אם ניתוק הגב כולו venעור עכבר tral, לעשות חתך אנכי באמצע העכבר בחזרה ואז לחתוך לאורך גבולות האזורים המגולחים סביב גוף העכבר. </li><li style=";text-align:right;direction:rtl"> בעדינות להפריד את העור מפני הצפק ושרירי גב, לשמירת דגימת העור עדיין עם המלקחיים תוך הפרדה בין מדגם העור בקצות הטו הסגורות של מספרי כירורגיות או אזמל. </li></ol></li><li style=";text-align:right;direction:rtl"> הכינו צלחת תרבית תאים 100 מ&quot;מ המכיל 10 מ&quot;ל של PBS קר כקרח. </li><li style=";text-align:right;direction:rtl"> לחסל שומן תת עורי על ידי השריית מדגם העור ב- PBS בצלחת מוכן בשלב 1.3.3 וקרצוף מדגם העור עם מלקחיים. </li></ol></li><li style=";text-align:right;direction:rtl"> זָנָב: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את הזנב של העכבר. </li><li style=";text-align:right;direction:rtl"> ביצוע חתך אנכי על הזנב עם מספריים כירורגיות או אזמל כירורגי, החל מבסיס הזנב ולהגיע אל קצה הזנב. </li><li style=";text-align:right;direction:rtl"> שימוש במלקחיים כדי לנתק את העור מהזנב. תפוס את הקצה לחתוך בבסיס הזנב בעדינות למשוך לכיוון הקצה תוך כדי לחיצה עלגוף של הזנב עם מלקחיים. </li><li style=";text-align:right;direction:rtl"> הסר את כל עודפי שומן על ידי גירוד מדגם העור בעדינות. </li></ol></li><li style=";text-align:right;direction:rtl"> שׁוֹדֵד דְרָכִים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חותכים את הרגל העכבר כולו עם מספריים כירורגיות. הימנעות חיתוך כל עור שעיר. </li><li style=";text-align:right;direction:rtl"> חותכים את העור של כף הרגל longitudinally מן הקרסול לתחילת הספרות. </li><li style=";text-align:right;direction:rtl"> החזק את עצמות קרסול עם מלקחיים או ביד בעת גרירת העור עם מלקחיים למשוך את העור כלפי הספרות כאילו הוא ממריא כפפה. </li></ol></li></ol> עיכול 2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את קוקטייל עיכול מפתרון המניות (שהוכן כמו רשימת חומרים) עם המתכון הבא: תערובת אנזימים (ראה רשימת חומרים עבור מפרטים) 300 מ&quot;ל / מיקרוגרם, DNAse1 50 U / ml. לדלל ב FBS 5% RPMI. </li><li style=";text-align:right;direction:rtl"> קוצצים דגימות עור עם מספריים לחתיכות קטנות לתוך צלחת פטרי 35 מ&quot;מ עם 2 מ&quot;ל של קוקטייל עיכול (לעור המטען: 2 מ&quot;ל של קוקטייל עיכול כל 2 ס&quot;מ 2 של העור, עד 3 מ&quot;ל סה&quot;כ). </li><li style=";text-align:right;direction:rtl">לדגור על 37 מעלות צלזיוס במשך 90 דקות. נער בעדינות את צלחת פטרי 1-2 פעמים במהלך הדגירה. </li><li style=";text-align:right;direction:rtl"> יוצקים דגימות עור מתעכל על מסננת 70 מיקרומטר תא 66 צלחת מ&quot;מ פטרי המכילה 1 מ&quot;ל של RPMI1640 FBS 5%. </li><li style=";text-align:right;direction:rtl"> שטפו את המסננת עם 5 מ&quot;ל של RPMI 1640 5% FBS. </li><li style=";text-align:right;direction:rtl"> סוחט את המדגם דרך מסננת התא עם בוכנה במזרק. </li><li style=";text-align:right;direction:rtl"> הבוכנה סומק, צלחת פטרי מסננת עם 20 מ&quot;ל של RPMI1640 FBS 5%, להעביר לתוך שפופרת 50 מ&quot;ל. </li></ol> 3. נוגדנים תיוג <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו את ההשעיה השיג פעמיים עם 5 מ&quot;ל של 1% PBS FBS (או BSA). </li><li style=";text-align:right;direction:rtl"> לייבל השעית התא שהושגה עם הנוגדנים הרצויים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Resuspend דגימות 100 μl / מדגם של תערובת המכילה מ&quot;ל 2 מיקרוגרם / של aCD16 / CD32 (שיבוט 2.4G2) מדולל FBS 1% PBS. </li><li style=";text-align:right;direction:rtl"> דגירה במשך 15 דקות ב 4 ° C על מנת לחסום את הלא ספציפי המחייב של הנוגדנים הרצויים באמצעות קולטני Fc. </li><li style=";text-align:right;direction:rtl"> מוסיף את הנוגדנים מצוינים בטבלה1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להכין תערובת דילול נוגדנים הרצוי 100 μl / מדגם של FBS 1% PBS בריכוז של 4 מיקרוגרם / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוספת 100 μl של תמהיל מוכן בשלב 3.2.3.1 לטעום כל כך בצינור, יהיו בהיקף של 200 μl של תערובת נוגדנים בריכוז סופי של 2 מיקרוגרם / מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> דגירה במשך 30 דקות ב 4 ° C ו לכסות מפני אור. </li></ol></li><li style=";text-align:right;direction:rtl"> לשטוף עם 1 מ&quot;ל של FBS 1% PBS, צנטריפוגות במשך 5 דקות ב 400 XG, וזורקים supernatant ו resuspend ב 300 μl של FBS 1% PBS. </li><li style=";text-align:right;direction:rtl"> הוסף DAPI בריכוז סופי של 2 מיקרוגרם / מ&quot;ל ​​ו דגירה על 4 מעלות צלזיוס, מוגן מפני אור במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> ניתוח הדגימות מתקבלות על ידי FACS. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> על מנת לבודד לויקוציטים, להשתמש באסטרטגיה gating הבאה: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ראשית לחסל את כל תאים מתים על ידי gating על אוכלוסיית DAPI-. </li><li style=";text-align:right;direction:rtl"> Singlets בחר בעלילה FSC-A לעומת FSC W כמצוין באיור 1 </strong&gt;. </li><li style=";text-align:right;direction:rtl"> שער התאים המבוססים על גודל גרעיניות טיפוסי של לויקוציטים עניין. </li><li style=";text-align:right;direction:rtl"> בחר CD45 + תאים בעלילה FSC-A CD45 כמצוין באיור 1. </li></ol></li><li style=";text-align:right;direction:rtl"> להבחין בין אוכלוסיות שונות של לויקוציטים ידי הביטוי של סמנים משטח כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זהה DCs כמו CD11c + MHC II + תאים. אלו ניתן לנתח לאחר מכן על מנת לתת ביטוי CD207 להבחין בין תאי לנגרהנס ואת CD207 + Dermal DCs מ CD207- Dermal DCs. </li><li style=";text-align:right;direction:rtl"> זהה המקרופאגים כמו CD64 + F4 / 80 + תאים. </li><li style=";text-align:right;direction:rtl"> לזהות תאי B כמו CD19 + MHCII + תאים. </li><li style=";text-align:right;direction:rtl"> לזהות תאי T כמו CD8 + או CD4 + תאים. </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.

תארנו שיטה להכנת תא בודדת השעיות מאזורי עור עכבר שונים. השיטה של ​​עיכול אמצנו, לא רק נותנת תשואות גבוהות אלא גם משמרת את הביטוי של סמני משטח, שהוא יסוד לניתוח FACS הבא. שימוש קוקטייל של collagenase I ו- II ו thermolysin, מבטיח יצווה מינימום להבד?...

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><tbody><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Supplemented RPMI 1640 medium&amp;nbsp;</td><td></td><td></td><td>RPMI1640 supplemented w/ L-glu, pen-strep w/o beta mercapto ethanol</td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>FBS</td><td></td><td></td><td></td></tr><tr><td>Liberase TM (Enzyme Mix)</td><td>Roche</td><td>5401119001</td><td>resuspend [2.5&amp;nbsp;mg/ml] in dH&lt;sub&gt;2&lt;/sub&gt;O; store aliquots at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dnase I</td><td>Sigma&amp;nbsp;</td><td>D4263-1VL</td><td>resuspend in RPMI 1640 w/o serum [1,000&amp;nbsp;U/ml]</td></tr><tr><td colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td>Surgical Scissors</td><td></td><td></td><td></td></tr><tr><td>Surgical Forceps</td><td></td><td></td><td></td></tr><tr><td>Surgical Scalpel</td><td></td><td></td><td></td></tr><tr><td>35 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>66 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>100 mm Petri dishes</td><td></td><td></td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainers</td><td>BD</td><td>352350</td><td></td></tr><tr><td>&lt;strong&gt;Digestion cocktail&lt;/strong&gt;</td><td></td><td></td><td>Dilute in RPMI 5% FBS</td></tr><tr><td>LIberase TM</td><td></td><td></td><td>300 &amp;micro;g/ml</td></tr><tr><td>DNAse1</td><td></td><td></td><td>50 U/ml</td></tr><tr><td colspan="4">&lt;strong&gt;Antibodies&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Clone&lt;/strong&gt;</td><td>&lt;strong&gt;Label&lt;/strong&gt;</td></tr><tr><td>aCD45.2</td><td></td><td>104</td><td>PE-Cy5.5</td></tr><tr><td>aCD11c</td><td></td><td>N418</td><td>PE-Cy7</td></tr><tr><td>CD11b</td><td></td><td>M1/70</td><td>FITC</td></tr><tr><td>F4/80</td><td></td><td>A 3-1</td><td>APC-Cy7</td></tr><tr><td>aCD4</td><td></td><td>Gk1.5</td><td>APC-Cy7</td></tr><tr><td>aCD8</td><td></td><td>53-6.7</td><td>PE</td></tr><tr><td>aCD64</td><td></td><td>X54-5/7.1</td><td>PE-Cy7</td></tr><tr><td>aCD207</td><td></td><td>4C7</td><td>APC</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.

עור מאזור אחר של הגוף היה מתעכל פי הפרוטוקול המתואר ומוכתם נוגדנים המצוינים. אסטרטגית gating מתוארת באיור 1. ראשית בחרו תאי חיים (תאי DAPI-), אז שער על singlets (FSC-A לעומת-W FSC) ועל תאים עם מורפולוגיה הלימפוציטים (FSC-A לעומת SSC-A). כאשר הדבר מתבקש, CD45 + ת...

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.

הכנת תא בודד השעיות עבור ניתוח Cytofluorimetric מאזורים העור עכבר שונים

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

18.2K Views.  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. 

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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

עכברי Dermal פיברובלסטים בידוד על ידי FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

בידוד תאי גזע זקיק שיער עוריות קרטינוציטים מן עור עכבר הגבה

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

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.

בידוד לימפוציטים מעור אדם עבור ניתוח פנוטיפי<em&gt; Ex Vivo</em&gt; תרבית תאים

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

בידוד תאי לוויין משרירי גפיים של עכבר וניתוח ציטומטריית זרימה

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

מחבר זרקור: ניתוח Cistrome בתאי גזע שריר עכבר 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

פרוטוקול יעיל לניתוח CUT&amp;RUN של תאי לוויין עכבר מבודדים ב-FAC

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

בידוד של פיברובלסטים נורמלים והסרטן הקשורים מרקמות טריות על ידי מיון תא הופעל פלואורסצנטי (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Biology

Preparing Mouse Thymic Epithelial Cells for Analysis of Intracellular Molecules

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the thymic cortex induce early T cell development and positive selection of cortical thymocytes. In contrast, medullary TECs (mTECs) in the thymic medulla attract positively selected thymocytes from the cortex and establish self-tolerance in T cells. A variety of molecules, including DLL4 and beta5t expressed in cTECs, as well as Aire and CCL21 expressed in mTECs, contribute to thymus function supporting T cell development and selection. Flow cytometric analysis of functionally relevant molecules in cTECs and mTECs is useful to improve our understanding of the biology of TECs, even though current methods for the preparation of single-cell suspensions of TECs can retrieve only a small fraction of TECs (approximately 1% for cTECs and approximately 10% for mTECs) from young adult mouse thymus. Because many of these functionally relevant molecules in TECs are localized within the cells, we describe our protocols for the preparation of single-cell suspension of mouse TECs and the staining of intracellular molecules for flow cytometric analysis.

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric AnalysisMechanisms

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

Thymic epithelial cells (TECs) play an essential role in promoting the development and repertoire selection of T cells. Cortical TECs (cTECs) in the ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

פרוטוקול מעוקב מדמיין ביטוי החלבון ברזולוציה תא יחיד תכשירים לעור הר שלם

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

ניתוח של המתלים תא מבודד מרקמות Solid ידי זרימת cytometry ספקטרלי

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

הכנת תא בודד השעיות עבור ניתוח Cytofluorimetric מאזורים העור עכבר שונים

Summary

Explore More Videos

Chapters in this video

עכברי Dermal פיברובלסטים בידוד על ידי FACS

בידוד תאי גזע זקיק שיער עוריות קרטינוציטים מן עור עכבר הגבה

בידוד לימפוציטים מעור אדם עבור ניתוח פנוטיפי

פרוטוקול יעיל לניתוח CUT&RUN של תאי לוויין עכבר מבודדים ב-FAC

פרוטוקול יעיל לניתוח CUT&RUN של תאי לוויין עכבר מבודדים ב-FAC

פרוטוקול יעיל לניתוח CUT&RUN של תאי לוויין עכבר מבודדים ב-FAC

בידוד של פיברובלסטים נורמלים והסרטן הקשורים מרקמות טריות על ידי מיון תא הופעל פלואורסצנטי (FACS)

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis

פרוטוקול מעוקב מדמיין ביטוי החלבון ברזולוציה תא יחיד תכשירים לעור הר שלם

ניתוח של המתלים תא מבודד מרקמות Solid ידי זרימת cytometry ספקטרלי