The authors would like to thank to Marilyn Dietrich at the LSU Flow Cytometry Core Facility for her help with the cell sorting and Peter Mottram for his assistance with the microphotographs. This work was funded by the Flight Attendant Medical Research Institute, the LSU-Competitive Research Program Award, and the NIH/NIAID Grants P20 RR020159 and R03AI081171.

1. Lung perfusion and single cell suspension
<ol>
 <li>Euthanize the mouse with an intraperitoneal injection of ketamine/xylazine anesthetic mixture (in mg/mouse ketamine 1.8, xylazine 0.19) and exsanguination via the femoral vein</li>
 <li>Expose the thoracic cavity by cutting and gently pulling back the outer skin of the peritoneum. Proceed to open the diaphragm by cutting the rib cage to expose both the heart and lungs.</li>
 <li>Perfuse gently the lungs using a 5 ml syringe filled with EDTA-HBSS (1mM EDTA in calcium/Magnesium-free HBSS). Connect a 25 G needle and insert needle into the right ventricle. To prevent leakage during injection, secure the myocardiac tissue around the needle by using forceps. Gently inject solution while maintaining a constant pressure. Accurate perfusion will result in lung inflation and a color change to pink/white.</li>
 <li>Remove draining lymph nodes to discard any potential contamination from this tissue.</li>
 <li>Collect lung tissue, transfer to a petri dish on ice, and chop it to small pieces using a razor blade.</li>
 <li>Transfer grounded tissue into a gentleMACS C tube containing 5 ml/lung of collagenase digestion solution (collagenase type 1A 0.5 mg/ml plus type IV bovine pancreatic DNase 20 &mu;g/ml in HBSS containing 5% FBS, 100 U/ml penicillin and 100 &mu;g/ml streptomycin).</li>
 <li>Use the gentleMACS dissociator to obtain a single cell suspension. Choose program: m_lung_01. Incubate sample at 37 &deg;C for 30 minutes. Shake tube every 5 minutes to resuspend tissue fragments. Proceed with program m_lung_02.</li>
 <li>Transfer sample to a 15 ml conical tube and centrifuge for 10 minutes at 335 x g at 4 &deg;C. After this step, keep all reagents and centrifugations at 4 &deg;C to prevent reduction in cell viability.</li>
 <li>Discard supernatant and lyse remaining red blood cells by adding 2 ml of ACK lysing buffer for 1 minute at room temperature. Wash cells with 13 ml of cold PBS/0.5% BSA and centrifuge for 10 minutes at 335 x g at 4&deg;C.</li>
 <li>Discard supernatant, resuspend total cells in 5 ml of cold PBS/0.5%BSA and pass thorough a 100 &mu;m nylon mesh.</li>
 <li>Determine the total cell number by using trypan blue exclusion dye.</li>
</ol>
2. Magnetic isolation and CD11c+ cells enrichment
<ol>
 <li>Centrifuge single cell suspension for 10 minutes at 200 x g at 4 &deg;C and discard supernatant.</li>
 <li>Resuspend cell pellet in 400 &mu;l of separation buffer (PBS, 0.5% BSA, and 2 mM EDTA) per 108 total cells. Block Fc-mediated unspecific antibody binding by using anti-CD16/CD32 antibody (0.5&mu;g/ 106 cells) for 30 minutes at 4 &deg;C.</li>
 <li>Wash cells with 10 ml of cold separation buffer and centrifuge for 10 minutes at 200 x g at 4 &deg;C. Resuspend cells in 400 &mu;l of separation buffer.</li>
 <li>Add 100 &mu;l of CD11c MicroBeads per 108 total cells. Mix well and incubate for 15 minutes in the refrigerator (2-8 &deg;C).</li>
 <li>Wash cells with 10 ml of cold separation buffer and centrifuge for 10 minutes at 200 x g at 4 &deg;C and discard supernatant.</li>
 <li>Resuspend the cell pellet in 3 ml of cold separation buffer.</li>
 <li>Perform magnetic separation using the autoMACS ProTM Separator by selecting the program posseld. Collect the positive fraction (0.5 ml).</li>
</ol>
3. Conventional dendritic cell (cDC) isolation
<ol>
 <li>Incubate enriched CD11c positive cell suspension with anti-CD11c PE-Cy7 and anti-I-A/I-E (MHC-II)-FITC antibodies for 30 minutes at 4 &deg;C. Optimized concentration of antibodies is 0.5 &mu;g/ 106 of cells.</li>
 <li>Wash cells with cold PBS/0.5% BSA and centrifuge for 10 minutes at 335 x g at 4 &deg;C. Resuspend cells in 0.5 ml of PBS/0.5% BSA and pass them through a 40 &mu;m nylon mesh.</li>
 <li>Proceed to sort the cells by using the FACS Aria cell sorter in the purity mode. Add 100 &mu;l of PBS/0.5% BSA to the collection tubes in order to prevent damage of purified cells. Keep your cells at a refrigerated temperature throughout the cell sorting process.</li>
</ol>
4. Alternate protocol for obtaining a single cell suspension and CD11c+ cells enrichment
<ol>
 <li>In replacement of the gentleMACS dissociator, grounded lung tissue from step 1.5 can be transferred to a 15 ml conical tube containing 5 ml of collagenase solution per lung and incubate for 1 hour at 37 &deg;C. Vortex cells every 15 minutes in order to resuspend tissue fragments. Disrupt the tissue by passing the sample 6-8 times in and out through a 3 ml syringe connected to a 20 G needle. Avoid making bubbles during the process, otherwise the cell viability will be compromised. Once single cell suspension is obtained, continue to step 1.8.</li>
 <li>In replacement of the AutoMACSProTM separator, magnetic isolation for the enrichment of the CD11c cells, can also be performed by passing the cell suspension manually through two MACS columns. The selection of the appropriate columns will vary depending of the sample size.</li>
</ol>
5. Representative Results:
Lung cDC are identified as CD11chi/MHC-IIhi cell population. As shown in Fig. 1, cDC represent a smaller percentage of 1.4% when compared to some other tissues such as spleen (4.8%). However, in contrast to spleen, lung CD11c positive cells express different amounts of MHC-II, including a cell population different than that of cDC. After the single-cell preparation, the cell yield of total lung cells was about 3.0 to 3.8 x 107 cells/lung with a viability of 60-70 % from which a little over 1% represented cDC subset. This percentage was increased after the CD11c magnetic isolation where the total lung cDC was incremented about 10 times (&gt;16%) from the original preparation (Fig. 2A). However, CD11c cells expressing low amounts of MHC-II (CD11chi/MHC-IIlo, macrophages) represented a high contaminating cell population (&gt;70%) which was mixed with the lung cDC subset. As shown in Fig. 2B, those cells were eliminated after the cell sorting step when the cDC purity reached &gt;96%. The usual yield of cDC after the whole procedure was 5 x 104 cells/lung. Microphotographs show morphological characteristics of cDC as rounded cells with the typical dendrites (upper panel). On the other hand, CD11chi/MHC-IIlo represented a morphologically different subset that shows cellular protuberances which are typical of macrophages (M&Phi;, lower panel) 12.
<img alt="Figure 1" src="/files/ftp_upload/3563/3563fig1.jpg" /> 
Figure 1. Differential DC frequency in mouse lung and spleen. Lung and spleen tissue from C57BL/6 mice were collected and treated with collagenase. Following collagenase digestion, cells were stained with anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Representative flow cytometry plots show percentages of cDC (CD11chi/MHC-IIhi) in lung and spleen.
<img alt="Figure 2" src="/files/ftp_upload/3563/3563fig2.jpg" /> 
Figure 2. Isolation of cDC from mouse lung. Mouse lung single cell suspension was labeled with anti-CD11c microbeads and passed through an automatic cell separator. A) A representative plot shows the percentages of cDC (CD11chi/MHC-IIhi) and Macrophages (M&Phi;, CD11chi/MHC-IIlo) populations after CD11c enrichment. Further staining of the enriched fraction followed using anti-mouse CD11c-PE-Cy7 and anti-mouse I-A/I-E-FITC. Double positive cells were sorted and analyzed by flow cytometry. To identify their morphology, cells were cytospun and stained with a modified Wright-Giemsa staining. B) Representative plots show percentages of cDC and M&Phi; after cell sorting. Microphotographs show a representative image of lung cDC and lung M&Phi;. Scale bar = 20 &mu;m.

<ol>
	<li>Pulendran, B., Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+pathogens+and+tuning+immune+responses.">Sensing pathogens and tuning immune responses.</a> Science. 293, 253-256 (2001).</li><li>Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Manicassamy, S., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+control+of+tolerogenic+responses.">Dendritic cell control of tolerogenic responses.</a> Immunol. Rev. 241, 206-227 (2011).</li><li>Steinman, R. M., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+cell+type+in+peripheral+lymphoid+organs+of+mice.+I.+Morphology,+quantitation,+tissue+distribution.">Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.</a> J. Exp. Med. 137, 1142-1162 (1973).</li><li>Asselin-Paturel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+type+I+IFN-producing+cells+are+immature+APCs+with+plasmacytoid+morphology.">Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.</a> Nat. Immunol. 2, 1144-1150 (2001).</li><li>Chan, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-producing+killer+dendritic+cells+provide+a+link+between+innate+and+adaptive+immunity.">Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity.</a> Nat. Med. 12, 207-213 (2006).</li><li>Taieb, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+dendritic+cell+subset+involved+in+tumor+immunosurveillance.">A novel dendritic cell subset involved in tumor immunosurveillance.</a> Nat. Med. 12, 214-219 (2006).</li><li>Guerrero-Plata, A., Kolli, D., Hong, C., Casola, A., Garofalo, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+pulmonary+dendritic+cell+function+by+paramyxovirus+infections.">Subversion of pulmonary dendritic cell function by paramyxovirus infections.</a> Journal of Immunology. 182, 3072-3083 (2009).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol. Rev. 114, 181-217 (1990).</li><li>Sung, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+lung+CD103+(alphaE)-beta7+integrin-positive+epithelial+dendritic+cell+population+expressing+Langerin+and+tight+junction+proteins.">A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.</a> J. Immunol. 176, 2161-2172 (2006).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+CD11c++MHC+class+II-+precursors+generate+lung+dendritic+cells+during+respiratory+viral+infection,+but+are+depleted+in+the+process.">Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process.</a> J. Immunol. 177, 2536-2542 (2006).</li><li>Bhatia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+of+alternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype.</a> PLoS One. 6, e15943-e15943 (2011).</li><li>Shao, Z., Makinde, T. O., McGee, H. S., Wang, X., Agrawal, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fms-like+tyrosine+kinase+3+ligand+regulates+migratory+pattern+and+antigen+uptake+of+lung+dendritic+cell+subsets+in+a+murine+model+of+allergic+airway+inflammation.">Fms-like tyrosine kinase 3 ligand regulates migratory pattern and antigen uptake of lung dendritic cell subsets in a murine model of allergic airway inflammation.</a> J. Immunol. 183, 7531-75381 (2009).</li><li>Hao, X., Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+respiratory+dendritic+cell+subsets+to+influenza+virus+infection.">Differential response of respiratory dendritic cell subsets to influenza virus infection.</a> J. Virol. 82, 4908-4919 (2008).</li></ol>

The authors have nothing to disclose.

Isolation of pulmonary mouse DC is an important technique for the study of a wide range of respiratory stimuli. The process of obtaining these cells includes critical steps that prevent loss of cells as well as cell viability and purity. Perfusing the lung before collection will help to eliminate any peripheral cells as well as reduce contaminant erythrocytes. The use of the automated dissociation can be advanteogus when a large number of lungs are handled, otherwise you can opt for the alternative protocol, keeping in m...

<table width="200" border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>ACK lysing buffer</td>
 <td>Invitrogen</td>
 <td>D6-0005DG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-mouse CD11c (HL3)</td>
 <td>BD Pharmingen</td>
 <td>5580979</td>
 <td>PE-Cy7 conjugated</td>
 </tr>
 <tr>
 <td>Anti-mouse I-A/I-E (269)</td>
 <td>BD-Pharmingen</td>
 <td>553623</td>
 <td>FITC conjugated </td>
 </tr>
 <tr>
 <td>Collangenase Type 1A</td>
 <td>Sigma</td>
 <td>9891-500MG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell strainers</td>
 <td>BD Falcon</td>
 <td>352340, 352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11c (N418) Microbeads</td>
 <td>Miltenyi</td>
 <td>130-052-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>D5025-150KU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank&rsquo;s Balanced Salt solution</td>
 <td>Invitrogen</td>
 <td>14170</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes buffer solution</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Petri dishes 60 mm</td>
 <td>BD Falcon</td>
 <td>351016</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GentleMACS&trade; C tubes</td>
 <td>Miltenyi</td>
 <td>130-093-237</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gentle MACS dissociator</td>
 <td>Miltentyi</td>
 <td>130-093-235</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AutoMACS-Pro&trade;</td>
 <td>Miltenyi</td>
 <td>130-092-545</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FASCS Aria</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

isolation of mouse lung dendritic cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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Immunology and Infection

28.9K 次观看.  Louisiana State University. 描述一个高度纯化的小鼠肺树突状细胞的准备。具体的重点是给传统的树突状细胞亚群的隔离。

视频: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

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

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

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...