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.

Study approval: The experimental protocols were approved by the Italian Ministry of Health (Rome, Italy) according to the Decreto legislativo 27 gennaio 1992, n. 116 &#34;Attuazione della Direttiva n. 86/609/CEE in materia di protezione degli animali utilizzati a fini sperimentali o ad altri fini scientifici.&#34;
1. Obtaining Skin Samples
<ol>
	<li>Euthanize the mouse by cervical dislocation.</li>
	<li>Ear:
	<ol>
		<li>Cut out the hairless part of the ears of the mouse.</li>
		<li>Separate dorsal and ventral side by pulling them apart with forceps. Remove any remaining cartilages by gently scraping the inner part of the ears with forceps.</li>
	</ol>
	</li>
	<li>Trunk skin:
	<ol>
		<li>Shave the whole skin of the animal both with an electric razor and with delicate depilatory lotion.</li>
		<li>Cut off the desired skin sample (from 2 cm2 up to the whole animal skin).
		<ol>
			<li>If cutting off the entire dorsal and ventral mouse skin, make a vertical cut in the middle of the mouse back and then cut along the borders of the shaved areas around the mouse body.</li>
			<li>Gently separate the skin from the peritoneum and back muscles, keeping the skin sample still with the forceps while separating the skin sample with the closed round edged tips of a surgical scissors or a scalpel.</li>
		</ol>
		</li>
		<li>Prepare a 100 mm cell culture plate containing 10 ml of ice cold PBS.</li>
		<li>Eliminate subcutaneous fat by submerging the skin sample in PBS in the plate prepared in step 1.3.3 and scraping the skin sample with forceps.</li>
	</ol>
	</li>
	<li>Tail:
	<ol>
		<li>Cut off the tail of the mouse.</li>
		<li>Make a vertical cut on the tail with a surgical scissors or surgical scalpel, starting from the tail base and reaching to the tip of the tail.</li>
		<li>Use forceps to detach the skin from the tail. Grab the edge of the cut at the base of the tail and gently pull toward the tip while holding the body of the tail with forceps.</li>
		<li>Remove any excess fat by gently scraping the skin sample.</li>
	</ol>
	</li>
	<li>Footpad
	<ol>
		<li>Cut out the entire mouse foot with surgical scissors. Avoiding cutting any hairy skin.</li>
		<li>Cut the skin of the foot longitudinally from the ankle to the beginning of the digits.</li>
		<li>Hold the ankle bones with forceps or by hand while grabbing the skin with forceps and pull the skin toward the digits as if taking off a glove.</li>
	</ol>
	</li>
</ol>
2. Digestion
<ol>
	<li>Prepare the digestion cocktail from the stock solution (prepared as in Materials List) with the following recipe: Enzyme mix (see Materials List for specifications) 300 &#956;g/ml, DNAse1 50 U/ml. Dilute in RPMI 5% FBS.</li>
	<li>Chop skin samples with scissors into small pieces into a 35 mm Petri dish with 2 ml of Digestion Cocktail (For trunk skin: 2 ml of Digestion Cocktail every 2 cm2 of skin, up to 3 ml total).</li>
	<li>Incubate at 37 &#176;C for 90 min. Gently shake the Petri dish 1-2 times during incubation.</li>
	<li>Pour digested skin samples on a 70 &#956;m cell strainer in 66 mm Petri dish containing 1 ml of RPMI1640 5% FBS.</li>
	<li>Wash the strainer with 5 ml of RPMI 1640 5% FBS.</li>
	<li>Squeeze the sample through the cell strainer with a syringe plunger.</li>
	<li>Flush plunger, Petri dish and strainer with 20 ml of RPMI1640 5% FBS, transfer into 50 ml tube.</li>
</ol>
3. Antibodies and Labeling
<ol>
	<li>Wash the obtained suspension twice with 5 ml of PBS 1% FBS (or BSA).</li>
	<li>Label the obtained cell suspension with the desired antibodies.
	<ol>
		<li>Resuspend the samples in 100 &#956;l/sample of a mix containing 2 &#956;g/ml of aCD16/CD32 (clone 2.4G2) diluted in PBS 1% FBS.</li>
		<li>Incubate for 15 min at 4 &#176;C in order to block non-specific binding of the desired antibodies via Fc receptors.</li>
		<li>Add the antibodies indicated in Table 1.
		<ol>
			<li>Prepare a mix diluting the desired antibodies in 100 &#956;l/sample of PBS 1% FBS at a concentration of 4 &#956;g/ml.</li>
			<li>Add 100 &#956;l of the mix prepared in step 3.2.3.1 to each sample so that in the tube, there will be a volume of 200 &#956;l of antibody mix at a final concentration of 2 &#956;g/ml.</li>
		</ol>
		</li>
		<li>Incubate for 30 min at 4 &#176;C and cover from light.</li>
	</ol>
	</li>
	<li>Wash with 1 ml of PBS 1% FBS, centrifuge for 5 min at 400 x g, discard the supernatant and resuspend in 300 &#956;l of PBS 1% FBS.</li>
	<li>Add DAPI at a final concentration of 2 &#956;g/ml and incubate at 4 &#176;C, protected from light for 10 min.</li>
	<li>Analyze the samples obtained by FACS.
	<ol>
		<li>In order to isolate leukocytes, use the following gating strategy:
		<ol>
			<li>First eliminate any dead cells by gating on the DAPI- population.</li>
			<li>Select singlets in the FSC-A vs. FSC W plot as indicated in Figure 1.</li>
			<li>Gate the cells based on size and granularity typical of the leukocytes of interest.</li>
			<li>Select CD45+ cells in the FSC-A CD45 plot as indicated in Figure 1.</li>
		</ol>
		</li>
		<li>Distinguish the different populations of leukocytes by the expression of surface markers as follows:
		<ol>
			<li>Identify DCs as CD11c+ MHC II+ cells. These can be later analyzed for the expression of CD207 to distinguish Langerhans cells and CD207+ Dermal DCs from CD207- Dermal DCs.</li>
			<li>Identify Macrophages as CD64+ F4/80+ cells.</li>
			<li>Identify B cells as CD19+ MHCII+ cells.</li>
			<li>Identify T cells as CD8+or CD4+ cells.</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. 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The authors declare that they have no competing financial interests.

We have described a method for the preparation of single-cell suspensions from different mouse skin regions. The method of digestion we have adopted, not only gives high yields but also preserves the expression of surface markers, which is fundamental for the subsequent FACS analysis.
The use of a cocktail of collagenase I and II and thermolysin, guarantees minimum batch to batch differences in enzyme activity making this method highly reproducible. Other published methods rely on different enzyme cocktails but in our hands they give lower yields and, more importantly, are less reproducible. Our method, however, does not discriminate between cells populating the epidermis and cells populating the dermis. Since in many cases it could be useful to distinguish between these two different compartments, we direct the reader to these previous works that use such methods.15
In our experience skin samples from the ear and tail are easier to digest than the trunk skin where the subcutaneous grease layer has to be thoroughly scraped in order to make the dermis accessible to the enzyme cocktail. The de-greasing step, though as well as the cutting should be performed quickly and gently in order to maintain cell viability. A prolonged or too thorough degreasing step can result in damage to the immune cells resident in the skin and therefore result in much lower yield and decreased viability of the recovered cells.
It should also be noted that to preserve the integrity of surface markers, the digestion should be performed in a medium devoid of reducing agents such as &#946;-mercaptoethanol. To avoid unwanted damage to surface markers, the incubation time should be kept as shorter as possible and, in any event, not prolonged for more than 90 min.
Particular care should be, also, taken when pouring the digested skin samples into the cell strainer. It is important to thoroughly wash the digested skin in order to wash away the liberated cells before smashing the digested sample with the syringe plunger. With this step the skin sample is washed from the liberated immune cells that then do not undergo any more physical stress. The smashing should also be performed gently in order not to kill any cells and both the cell strainer and the syringe plunger should be thoroughly washed in the subsequent washing step.
Skin digestion can be also performed in a 50 ml falcon in a water bath at 37 &#176;C. In this case the skin can be chopped beforehand and then put into the falcon tube. Note that the skin samples should always be submerged in digestion buffer during the incubation and that ear-pieces stick to the sides of falcon tubes. When using falcon tubes, therefore, make sure the pieces of chopped skin stay submerged in the digestion medium.
This method can be useful in many different experimental settings, particularly when studying non-inflamed skin. During inflammation, vasodilation and tissue swelling loosen the structure of the tissue and immune cells from blood are massively recruited to the skin. The efficiency of cell recovery is, thus, much higher. In homeostatic conditions, an effective way to digest the tight collagen matrix of the dermis is, in fact, needed in order to recover the immune cell constituting the complex network populating the skin.
The reader might also be interested in a comprehensive review that covers the origin and phenotype of different dendritic cells and macrophages populating the skin in both homeostatic and inflammatory conditions.16
In different experimental settings, different portions of mouse skin can be treated. For instance, in skin transplantation models, a portion of the tail could be transplanted on the trunk of a receiving animal; when studying edema formation or immunization procedures, stimuli are often injected on the footpad of mice, while the ear is frequently used to assess T cell memory with DTH assays. With our method we could efficiently extract skin resident immune cells from different locations making our method useful for the experimental setting of choice.

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.

Skin from different region of the body was digested according to the protocol described and stained with the indicated antibodies. The gating strategy is depicted in Figure 1. First select live cells (DAPI- cells), then gate on singlets (FSC-A vs. FSC-W) and on cells with lymphocyte morphology (FSC-A vs. SSC-A). When indicated, CD45+ cells from hematopoietic origin are selected.
DCs are labeled with anti-CD11c, -MHCII, and -CD45 antibodies, Macrophages are identified as F4/80+ CD64+ CD45+ cells, B cells as CD19+CD45+ MHCII+ cells, CD4+T lymphocytes as CD4+CD45+ cells, and CD8+ T lymphocytes as CD8+CD45+ cells (Figure 2) .
This method guarantees high viability of the digested population with more than 50% of total viable cells (DAPI-) in all regions of the skin that are analyzed (Figure 3A). An indication of the number of CD45+ cells yield per cm2 is depicted in Figure 3B.
An estimate of the total number of DCs, Macrophages, B cells and CD4+ or CD8+ T cells obtainable from the digestion of 1 cm2 of mouse skin is indicated in Figure 3C.
<img alt="Figure 1" src="/files/ftp_upload/52589/52589fig1.jpg" /> 
Figure 1. Identification of cells of hematopoietic origin in skin single-cell suspensions. Single-cell suspensions have been stained with DAPI and the anti-CD45 antibody. Among live cells (DAPI-negative), singlets (FSC-A vs FSC-H) are analyzed to select cells with leukocyte morphology (FSC-A vs. SSC-A). Cells of hematopoietic origin are identified as CD45+.
<img alt="Figure 2" src="/files/ftp_upload/52589/52589fig2.jpg" /> 
Figure 2. Analysis of immune cells present in the mouse skin derived from different regions. Cells are gated as indicated in Figure 1. DCs are identified as CD11c+MHC II+ CD45+ cells and later stained with CD207 to distinguish Langerhans cells and CD207+ dermal DCs from CD207- ; Macrophages are identified as CD64+F4/80+CD45+ cells; T lymphocytes are divided in CD8+CD45+ cells and CD4+CD45+ cells; B cells are identified as MHCII+CD19+CD45+ cells. Percentages are referred to the total CD45+ cells.
<img alt="Figure 3" src="/files/ftp_upload/52589/52589fig3.jpg" /> 
Figure 3. Quantitative analysis of the immune cells present in the mouse skin from different regions. (A) Percentage of viable (DAPI-) cells derived from the indicated regions of mouse skin. (B) Absolute numbers CD45+ cells in the different regions of mouse skin expressed in number of cells per cm2 of skin. (C) Estimate number of DCs, M&#934;, B cells and CD4+ or CD8+ T cells derived from 1 cm2 of skin from the indicated regions of mouse skin.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Staining</td>
			<td>Concentration</td>
			<td>Time</td>
			<td>Temperature</td>
		</tr>
		<tr>
			<td colspan="5">DCs Mix</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>PE-Cy7</td>
			<td>2 &#181;g/ml</td>
			<td>30 min</td>
			<td>4 &#176;C</td>
		</tr>
		<tr>
			<td>MHC II</td>
			<td>PE</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PE-Cy5.5</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD207</td>
			<td>APC</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td colspan="5">Macrophages Mix</td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>APC-Cy7</td>
			<td>2 &#181;g/ml</td>
			<td>30 min</td>
			<td>4 &#176;C</td>
		</tr>
		<tr>
			<td>MHC II</td>
			<td>PE</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PE-Cy5.5</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD64</td>
			<td>PE-Cy7</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td colspan="5">T and B cells Mix</td>
		</tr>
		<tr>
			<td>CD8a</td>
			<td>PE</td>
			<td>2 &#181;g/ml</td>
			<td>30 min</td>
			<td>4 &#176;C</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>APC-Cy7</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>APC</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>PE-Cy5.5</td>
			<td>&#34;</td>
			<td>&#34;</td>
			<td>&#34;</td>
		</tr>
	</tbody>
</table>
Table 1. Indications for the staining of the different populations of immune cells populating the skin. Three mixes are indicated for staining of DCs, Macrophages and lymphocytes.

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.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.1K 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

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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