The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

In this video publication and protocol, all animal procedures were conducted in accordance to the animal protocol approved by the Cantonal Authority Basel-Stadt, Switzerland.
1. Lymph Nodes Preparation and Digestion
<ol>
	<li>Pre-heat water in a beaker to 37 &#176;C on a magnetic stirrer with heating plate.</li>
	<li>Prepare Basic Medium as following: DMEM medium (without pyruvate) supplemented with 2% FCS, 1.2 mM CaCl2 and Pen/Strep (100 units of penicillin, 100 &#181;g of streptomycin).</li>
	<li>Sterilize all dissection instruments before use.</li>
	<li>Euthanize lymph node donor mice per CO2 asphyxiation and aseptically dissect the lymph nodes. Do not dissect the surrounding fat. NOTE: This protocol is optimized for peripheral skin-draining lymph node (inguinal, brachial, axillary).</li>
	<li>Place lymph nodes in a sterile Petri dish containing 2 ml ice cold basic medium.</li>
	<li>Disrupt the lymph node capsule using two 25 G needles fixed on 1 ml syringe.</li>
	<li>Transfer the disrupted lymph node tissue in a 5 ml polypropylene round-bottom tube containing 750 &#181;l basic medium supplemented with 1 mg/ml Collagenase IV and 40 &#181;g/ml DNAse I.</li>
	<li>Add one sterile magnetic stirrer in each tube.</li>
	<li>Place tube in the beaker with 37 &#176;C preheated water and stir the tubes at a slow rate (1 round/sec) for 30 min.</li>
	<li>Remove the tube from the magnetic stirrer with heating plate and let lymph node fragments settle.</li>
	<li>Carefully remove the supernatant enriched in &#34;non-stromal cell&#34;. NOTE: If the analysis of T, B, dendritic cells and CD45-gp38-CD31- is foreseen, save the &#34;non-stromal cells&#34; fraction.</li>
	<li>Wash remaining lymph node tissue once with 750 &#181;l basic medium. NOTE: This step is necessary only if working with immune-competent mice.</li>
	<li>Let lymph node fragments settle.</li>
	<li>Remove the non-stromal cell-floating fraction.</li>
	<li>Add to the lymph node fragments 750 &#181;l basic medium supplemented with 3.5 mg/ml Collagenase D and 40 &#181;g/ml DNase I.</li>
	<li>Place tube back in the beaker containing the 37 &#176;C preheated water.</li>
	<li>Digest lymph node tissue for 5 min while slowly stirring.</li>
	<li>Disaggregate lymph node tissue fragments by pipetting and mixing 700 &#181;l for 10 cycles at maximal speed using an automated multichannel pipette. NOTE: This disrupts lymph node tissue to improve digestion.</li>
	<li>Place the tube back in the beaker with 37 &#176;C preheated water.</li>
	<li>Digest lymph node tissue fragments for another 10 min while slowly stirring.</li>
	<li>Disaggregate lymph node tissue fragments by pipetting and mixing for 99 cycles at maximal speed using an automated multichannel pipette.</li>
	<li>Add 7.5 &#181;l of 0.5 M EDTA to ensure maintenance of single cell suspension.</li>
	<li>Disaggregate lymph node tissue fragments by pipetting and mixing for 99 cycles at maximal speed using an automated multichannel pipette.</li>
	<li>Add 750 &#181;l basic medium and pass cells through a 70 &#181;m nylon mesh.</li>
	<li>Centrifuge cell suspension 5 min at 1,500 x g, 4 &#176;C.</li>
	<li>Stromal cells can now be used for further analysis.</li>
</ol>
2. Staining of Lymph Node Stromal Cells
<ol>
	<li>Use a combination of anti-CD45, anti-podoplanin (gp38), anti-CD31 antibodies to successfully recognize TRC, LEC, BEC and DN cells.</li>
	<li>Incubate the digested lymph node tissue with Live/Dead tracker, anti-CD45-FITC (1:200), anti-gp38-PE (1:200), anti-CD31-APC (1:200) in 100 &#956;l HBSS containing 2% FCS for at least 20 min, 4 &#176;C, in the dark.</li>
	<li>Wash the cells adding 500 l of HBSS containing 2% FCS</li>
	<li>Centrifuge cell suspension 3 min at 1500 x g, 4&#176;C.</li>
	<li>Re-suspend in 100 &#956;l of HBSS containing 2% FCS.</li>
	<li>Run the stained cells in a flow cytometer equipped with the following optics: excitation source with up to three lasers: blue (488 nm, air-cooled, 20 mW solid state), red (633 nm, 17 mW HeNe), and violet (405 nm, 30 mW solid state).</li>
	<li>Gate CD45- cells to exclude hematopoietic cells.</li>
	<li>Gate singlets (FSC-W) and live cells (LIVE/DEAD tracker) to exclude doublets and dead cells.</li>
	<li>Plot gp38 vs. CD31 to visualize TRC (gp38+CD31-), LEC (gp38+CD31+), BEC (gp38-CD31+) and DN (gp38-CD31-) cells (see Figure 1).</li>
</ol>

<ol><li>Mueller, S. N., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphoid+stroma+in+the+initiation+and+control+of+immune+responses%5BTitle%5D)+AND+%22Immunological+reviews%22%5BJournal%5D)">Lymphoid stroma in the initiation and control of immune responses.</a> Immunological reviews. 224, 284-294 (2008).</li>
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<li>Luther, S. A., Tang, H. L., Hyman, P. L., Farr, A. G., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coexpression+of+the+chemokines+ELC+and+SLC+by+T+zone+stromal+cells+and+deletion+of+the+ELC+gene+in+the+plt%2Fplt+mouse%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (23), 12694-12699 (2000).</li>
<li>Braun, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Afferent+lymph-derived+T+cells+and+DCs+use+different+chemokine+receptor+CCR7-dependent+routes+for+entry+into+the+lymph+node+and+intranodal+migration%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration.</a> Nature Immunology. 12 (9), 879-887 (2011).</li>
<li>Fletcher, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducible+isolation+of+lymph+node+stromal+cells+reveals+site-dependent+differences+in+fibroblastic+reticular+cells%5BTitle%5D)+AND+%22Frontiers+in+immunology%22%5BJournal%5D)">Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.</a> Frontiers in immunology. 2, 35(2011).</li>
<li>Lukacs-Kornek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulated+release+of+nitric+oxide+by+nonhematopoietic+stroma+controls+expansion+of+the+activated+T+cell+pool+in+lymph+nodes%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes.</a> Nature Immunology. 12 (11), 1096-1104 (2011).</li>
<li>Siegert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblastic+reticular+cells+from+lymph+nodes+attenuate+T+cell+expansion+by+producing+nitric+oxide%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.</a> PLoS ONE. 6 (11), e27618(2011).</li>
<li>Khan, O., Headley, M., Gerard, A., Wei, W., Liu, L., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+T+cell+priming+by+lymphoid+stroma%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Regulation of T cell priming by lymphoid stroma.</a> PLoS ONE. 6 (11), e26138(2011).</li>
<li>Svensson, M., Maroof, A., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromal+cells+direct+local+differentiation+of+regulatory+dendritic+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Stromal cells direct local differentiation of regulatory dendritic cells.</a> Immunity. 21 (6), 805-816 (2004).</li>
<li>Onder, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+cell-specific+lymphotoxin-%CE%B2+receptor+signaling+is+critical+for+lymph+node+and+high+endothelial+venule+formation%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.</a> The Journal of experimental medicine. 210 (3), 465-473 (2013).</li>
<li>Fletcher, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymph+node+fibroblastic+reticular+cells+directly+present+peripheral+tissue+antigen+under+steady-state+and+inflammatory+conditions%5BTitle%5D)+AND+%22The+Journal+of+experimental+medicine%22%5BJournal%5D)">Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions.</a> The Journal of experimental medicine. 207 (4), 689-697 (2010).</li>
<li>Fletcher, A. L., Malhotra, D., Turley, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymph+node+stroma+broaden+the+peripheral+tolerance+paradigm%5BTitle%5D)+AND+%22Trends+in+Immunology%22%5BJournal%5D)">Lymph node stroma broaden the peripheral tolerance paradigm.</a> Trends in Immunology. 32 (1), 12-18 (2011).</li>
<li>Cohen, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymph+node-resident+lymphatic+endothelial+cells+mediate+peripheral+tolerance+via+Aire-independent+direct+antigen+presentation%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.</a> Journal of Experimental Medicine. 207 (4), 681-688 (2010).</li>
<li>Lee, J. -W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+antigen+display+by+lymph+node+stroma+promotes+T+cell+tolerance+to+intestinal+self%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self.</a> Nat Immunol. 8, 181-190 (2006).</li>
<li>Nichols, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletional+self-tolerance+to+a+melanocyte%2Fmelanoma+antigen+derived+from+tyrosinase+is+mediated+by+a+radio-resistant+cell+in+peripheral+and+mesenteric+lymph+nodes%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Deletional self-tolerance to a melanocyte/melanoma antigen derived from tyrosinase is mediated by a radio-resistant cell in peripheral and mesenteric lymph nodes.</a> J Immunol. 179, 993-1003 (2007).</li>
<li>Gardner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletional+tolerance+mediated+by+extrathymic+Aire-expressing+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Deletional tolerance mediated by extrathymic Aire-expressing cells.</a> Science. 321, 843-847 (2008).</li>
<li>Gardner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrathymic+Aire-expressing+cells+are+a+distinct+bone+marrow-derived+population+that+induce+functional+inactivation+of+CD4%2B+T+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Extrathymic Aire-expressing cells are a distinct bone marrow-derived population that induce functional inactivation of CD4+ T cells.</a> Immunity. 39, 560-572 (2013).</li>
<li>Magnusson, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+presentation+of+antigen+by+lymph+node+stromal+cells+protects+against+CD8+T-cell-mediated+intestinal+autoimmunity%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Direct presentation of antigen by lymph node stromal cells protects against CD8 T-cell-mediated intestinal autoimmunity.</a> Gastroenterology. 134, 1028-1037 (2008).</li>
<li>Tewalt, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+endothelial+cells+induce+tolerance+via+PD-L1+and+lack+of+costimulation+leading+to+high-level+PD-1+expression+on+CD8+T+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.</a> Blood. 120 (24), 4772-4782 (2012).</li>
</ol>

The authors declare they have no competing financial interests.

The study of lymph node stromal cells recently became a research focus due to the development of two published digestion protocols6,13. Both protocols are adequate to gain single lymph node stromal cells but differ in the use of digestion enzymes and the time of digestion. Since stromal cells and their surface markers are sensitive to enzymatic digestion and mechanical stress, an optimized protocol is required.
The isolation of viable lymph node stromal cells from freshly dissected ...

Lymph nodes are specialized compartments where adaptive immune responses against foreign and self-antigens are initiated and coordinated. The procedure presented here describes a short enzymatic digestion combined with automated mechanical pipetting to obtain lymph node single cell suspension and gain access to viable lymph node stromal cells that maintain the surface expression of several molecules.
Lymph node stromal cell form the scaffold of the lymph node and fulfill three major functions: first they filter body fluids to sample antigens, pathogens and their pathogen associated molecular pattern (PAMPs), as well as cytokines and danger associated molecular pattern (DAMPs) present in the body. Second, they attract and instruct antigen presenting cells (APC) and lymphocytes to interact and initiate adaptive immune responses; and third, they provide a structural environment for the homeostasis and differentiation of lymphocytes1-3. During inflammation lymph node stromal cells produce growth factors, cytokines and chemokines, adapt to swelling thereby organizing the interaction between dendritic cells (DCs), T-, and B- cells. The orchestration of immune responses is only possible due to the complex structural architecture formed by different stromal cell populations.
Lymph node stromal cells are CD45 negative cells and can be distinguished by the expression of CD31 or gp38 in fibroblastic and endothelial cells1-6. Gp38+CD31- defines T zone reticular cells (TRC, also known as FRC: fibroblastic reticular cells), gp38+CD31+ defines lymphatic endothelial cells (LEC), gp38-CD31+ defines blood endothelial cells (BEC). Further, characterization of the subpopulations revealed the existence of other lymph node stromal cells. Indeed, a small pericyte-like cell population was characterized within the gp38-CD31- population7. Therefore, adaptation of the isolation procedure is advantageous for identification and characterization of the functional properties of different lymph node stromal cells.
Before the development of lymph node stromal cells digestion protocols the study of lymph node stromal cells was limited to in situ observations using tissue section and microscopy. Nevertheless, structural and functional studies showed important characteristics of lymph node stromal cells. Lymph node stromal cells are associated with podoplanin, collagen and extracellular matrix (ECM) proteins to form a complex 3 dimensional structure called conduit system, which transports lymph and associated-low molecular mass proteins from the subcapsular sinus of the lymph node to the high endothelial venules in the T cells zone8. DCs are in close contact with stroma cells and may be observed protruding into the tubular conduit structure to sample fluid and detect antigens8. The interaction of lymph node stromal cells (TRCs and LECs) with DCs is mediated by the release and presentation of chemokines CCL21 and CCL199,10. CCL19 and CCL21 are recognized by the CCR7 receptor facilitating DCs and T cells to migrate to the lymph node T cell zone4,11. Despite using similar chemokines, DCs and T cells have different migration routes into the lymph nodes12. Later, using enzymatic digestion of the lymph node and isolation of pure lymph node stromal cells, functional studies were performed on the role of the different lymph node stromal cells and their ability to interact with DCs and T/B cells6,13. First, the crosstalk between IFN-&#947; producing effector T cells and lymph node stromal cells induces the production of the metabolite nitric oxide shown to dampen T cell responses and proliferation in the secondary lymphoid organs14-16. Second, lymph node stromal cells have been reported to support the differentiation of regulatory DC subsets via the production of IL-1017, and to modulate na&#239;ve T cell homeostasis via the production of IL-76,18. Third, TLR expression in lymph node stromal cells suggests that stromal cells are susceptible to signal derived from an infection or self-molecules released during tissue injury. Indeed, the treatment of lymph node stromal cells with the ligand of TLR3 poly(I:C) induces a modest upregulation of major histocompatibility complex class I expression and upregulation of co-inhibitory molecule PD-L1, but not of costimulatory molecules, resulting in dramatic changes in peripheral tissue antigens expression19. Several groups have shown lymph node stromal cells express peripheral tissue antigens and induce tolerance of self-reactive T cells19,21-27. Therefore, understanding the interactions between lymph node stromal cells and the other migratory and resident lymph node cells will help to find new target molecules to allow activation or suppression of immune responses during inflammation. Therefore, the implementation of the published enzymatic separation of the lymph node is needed.
Previously published protocols use different combinations of collagenase-based enzymatic digestion with low mechanical stress6,19,20. However, long incubations with digestion enzymes or the different combination of digestion enzyme might degrade various surface molecules required to analyze the activation status and to identify new lymph node stromal cells. Depending on the type of the stromal cell analysis, the Link Protocol or Fletcher Protocol might be more suited. In the described procedure, a slightly shorter enzymatic digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells. This procedure enables highly reproducible isolation and distinction of lymph node stromal cell populations with low variability and more than 95% viability. The freshly isolated lymph node stromal cells can be directly used for surface marker expression, protein analysis, and transcriptional studies, as well as establishment of stromal cells lines to perform functional assays in vitro.

<table><tbody><tr><td>DMEM</td><td>LifeTechnologies-Gibco</td><td>41965-039</td><td></td></tr><tr><td>FCS</td><td></td><td></td><td>Final concentration 2%</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>499609</td><td>Final concentration 1.2 mM</td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td></td><td>&gt;160 units per mg dry weight, use at final concentration of 1 mg/ml</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>use at 3.5 mg/ml</td></tr><tr><td>DNAse I</td><td>Roche&amp;nbsp;</td><td>11284932001</td><td>use at 40 &amp;micro;g/ml</td></tr><tr><td>Stiring magnets</td><td>FAUST</td><td></td><td>5 mm long-2 mm &amp;oslash;</td></tr><tr><td>Polystyrene round bottom tubes 5 ml</td><td>Falcon-BD Bioscience</td><td></td><td></td></tr><tr><td>Magnetic stirrer with heating function</td><td>IKA-RCT-standard</td><td>9720250</td><td></td></tr><tr><td>Petri dishes 100 mm, sterile</td><td>TPP</td><td>6223201</td><td></td></tr><tr><td>25 G needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>anti mouse CD45 Ab</td><td>Biolegend</td><td></td><td>Clone 30-F11</td></tr><tr><td>anti mouse CD11c Ab</td><td>Biolegend</td><td></td><td>Clone N418</td></tr><tr><td>anti mouse Podoplanin Ab</td><td>Biolegend</td><td></td><td>Clone 8.1.1</td></tr><tr><td>anti mouse CD31 Ab</td><td>Biolegend</td><td></td><td>Clone MEC13.3</td></tr><tr><td>&lt;br/&gt; Eppendorf Xplorer plus, Multichannel</td><td>Eppendorf</td><td>4861 000.821/830</td><td>1.250 &amp;micro;l max. volume</td></tr><tr><td>anti mouse CD140a</td><td>Biolegend</td><td></td><td>Clone APA5</td></tr><tr><td>anti mouse CD80</td><td>Biolegend</td><td></td><td>Clone 16-10A1</td></tr><tr><td>anti mouse CD40</td><td>Biolegend</td><td></td><td>Clone 1C10</td></tr><tr><td>anti mouse I-Ab</td><td>Biolegend</td><td></td><td>Clone AF6-120.1</td></tr><tr><td>anti mouse CD274 (PD-L1)</td><td>Biolegend</td><td></td><td>Clone 10F.9G2</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td><td>Invitrogen</td><td>L10119</td><td></td></tr></tbody></table>

isolation of murine lymph node stromal cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

The present protocol is a modified digestion protocol published by Link et al., 20076 with a shorter digestion time (45 min maximum) due to mechanical disaggregation with an automated multichannel pipette. In addition, the procedure is more standardized, minimizes degradation of surface markers on different lymph node stromal cells and allows the handling of more than one sample at the same time.
Collagenase IV and Collagenase D in Links protocol6 and current pro...

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Isolation of Murine Lymph Node Stromal Cells

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

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JoVE Journal

Immunology and Infection

31.3K Views.  University of Basel and University Hospital Basel. Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells. 

Video: Isolation of Murine Lymph Node Stromal Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biology

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Optimizing Stromal Cell Isolation from Thymus and Bone Marrow

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Author Spotlight: Advancing Hematopoietic Research Using Stromal Cell Isolation for Single Cell Sequencing

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolating Endothelial Cells from Murine Dermal Lymphatic Capillaries

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Author Spotlight: Magnetic Bead-Based Isolation of Murine Dermal Lymphatic Endothelial Cells

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...