Name: An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets (Video) | JoVE Journal
Uploaded: 2016-04-18T13:00:00.000+00:00
Duration: 9 min 9 s
Description: Learn a high-yield method for isolating mouse dendritic cell subsets using Flt-3 ligand expansion and magnetic bead separation. This protocol achieves >94% purity for functional immunology studies.

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This work was supported by NIH/NIAID grant AI45889 to S.A.P. Flow cytometry studies were carried out using FACS core facilities supported by the Einstein Cancer Center (NIH/NCI CA013330) and Center for AIDS Research (NIH/NIAID AI51519).

Animal experiments are done in accordance with approved guidelines from the institutional animal care and usage committee (IACUC). All procedures requiring sterility are performed in a biosafety cabinet.
1. Implantation of B16.Flt3L Melanoma in Mice
<ol>
	<li>Culture the Flt-3 expressing B16 melanoma cell line in T25 tissue culture flasks at a density of 0.5 x 106 cells/ml&#160;in complete DMEM medium with 10% CO2 at 37 &#176;C. Grow until the cells reach ~90% confluence (~20 hr).</li>
	<li>Harvest the cells by trypsin digestion. Aspirate cell culture media and wash the adherent cells with PBS. Add 5 ml&#160;of 0.05% trypsin and incubate for 5 min at 37 &#176;C. Add cold 20 ml&#160;complete DMEM medium (4 &#176;C) containing fetal calf serum (FCS) to quench the protease digestion. Lift the cells off by lightly tapping followed by gently pipetting up and down.</li>
	<li>Collect the cells by centrifugation at 300 x g for 10 min&#160;at 4 &#176;C. Count the cells using a hemocytometer or an automated cell viability counter. Resuspend to a density of 108 cells/ml&#160;in PBS.</li>
	<li>Implant mice (C57BL/6 or another histocompatible strain) with tumor cells by subcutaneous injection as described by Machholz et al.19 at the base of the neck with 100 &#181;l&#160;of cell suspension (107 cells).</li>
	<li>Allow the tumor to grow until palpable or visible (2 - 10 mm in diameter, usually 7 - 12 days) before sacrificing animals for harvesting organs.</li>
</ol>
2. Preparation of Splenic Single Cell Suspension from Tumor Bearing Mice
<ol>
	<li>Anesthetize mice with an overdose of isoflurane (adjust flow rate of isoflurane to &#62;5% and continue exposure at least 2 min&#160;after breathing stops). Sacrifice the Flt-3 melanoma tumor bearing mouse by cervical dislocation according to institutional guidelines for euthanasia.</li>
	<li>Disinfect the skin with 70% ethanol and harvest spleen using aseptic technique with the aid of sterile scissors 20.</li>
	<li>Place the spleen into a sterile Petri dish for transport to the biosafety cabinet. Perform all steps from here on under sterile conditions.</li>
	<li>Transfer spleen to a fresh Petri dish and wash with RPMI medium. Cut the spleen into small (~0.2 cm2) pieces using a scalpel. Incubate for 30 min at 37 &#176;C with 10 ml&#160;of collagenase/DNase solution.</li>
	<li>Pour the partially digested suspension of splenic tissue onto a 70 &#181;m cell strainer. Compress the remaining tissue fragments through the mesh of the strainer using a 5 ml&#160;syringe plunger.</li>
	<li>Wash the mesh filter with 10 ml&#160;of fresh medium and discard the filter. Centrifuge the suspension at 300 x g for 10 min&#160;at 4 &#176;C to pellet the cells.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 2 ml&#160;of RBC lysis buffer. Incubate at RT for 10 min. Quench the RBC lysis buffer by adding 10 ml&#160;of complete RPMI medium.</li>
	<li>Pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Resuspend in appropriate volume to achieve cell density of 108 cells/ml&#160;in cell sorting buffer (e.g., MACS) containing 20 &#181;g/ml&#160;of Fc-block antibody (2.4G2).</li>
</ol>
3. Purification of Plasmacytoid DCs, CD8&#945;Pos DCs and CD8&#945;Neg DCs from Splenic Single Cell Suspension
Note: Isolation of the splenic DC subsets from single cell suspension is a multi-step procedure as illustrated in Figure 1. Perform all the steps using pre-cooled buffer and an ice water bath to maintain temperature at 4 - 8 &#176;C.&#160;All reagent volumes in the following section of the protocol are calculated for an initial input of 200 &#181;l of splenic cell suspension at 108 cells/ml). This can be scaled up or down based on your need by changing the volume of cell suspension (always at a density of 1 X 108 cells/ml) and other reagents proportionately in the initial step (3.1.1 and 3.2.1). Adjust reagent volumes in all later steps accordingly. For example, if starting with 100 &#181;l of splenic cell suspension at 1 x 108/ml, use half the stated reagent volumes in all steps.
<ol>
	<li>Isolation of Plasmacytoid DCs (pDC)
	<ol>
		<li>Add 300 &#181;l&#160;of biotin-labeled antibody cocktail provided in the plasmacytoid DC isolation kit to 200 &#181;l&#160;of splenic cell suspension.</li>
		<li>Mix thoroughly and incubate in ice water bath for 15 min. Tap the tube every 3 min&#160;to keep cells suspended, and to maximize antibody binding.</li>
		<li>Add 12 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g&#160;for 5 min at 4 &#176;C. Aspirate supernatant to remove unbound biotinylated antibodies.</li>
		<li>Resuspend the cell pellet in 500 &#181;l&#160;of cell sorting buffer. Add 300 &#181;l&#160;of anti-biotin antibody-conjugated beads. Repeat step 3.1.2 and 3.1.3.</li>
		<li>Discard supernatant and resuspend the cell pellet in 2 ml&#160;of cell sorting buffer. Perform all steps from this point on at RT&#160;using pre-cooled buffers.</li>
		<li>Place a fresh magnetic activated cell sorting LS column in the magnetic stand. Wash and equilibrate column with 5 ml&#160;of cell sorting buffer.</li>
		<li>Pipette the cell suspension onto the column, applying it gently to the center of the surface of the column bed. Collect the flow through.</li>
		<li>Wash the column with 10 ml&#160;of cell sorting buffer. Collect this flow through and combine with the flow through from step 3.1.7. The pDCs are enriched in this column flow through (FT1).</li>
		<li>Pellet the cells from FT1 by centrifugation at 300 x g for 5 min at 4 &#176;C, resuspend in 2 ml&#160;cell sorting buffer and pass the cells through a fresh LS column by repeating steps 3.1.6 - 3.1.8. This use of two sequential columns yields enhanced purity of isolated cells.</li>
		<li>Pellet cells by centrifugation at 300 x g for 5 min at 4 &#176;C, and resuspend with 2 ml&#160;complete RPMI. Place on ice until needed.</li>
	</ol>
	</li>
	<li>Isolation of CD8&#945;Pos and CD8&#945;Neg DCs 
	Note: The first step in this procedure is depletion of B, pDC, T and NK cells.
	<ol>
		<li>Starting with the whole spleen cell suspension (1 ml of 108&#160;cells/ml obtained in step 2.8), add 100 &#181;l&#160;of biotin-conjugated antibody cocktail provided in the CD8&#945;Pos DC isolation kit.</li>
		<li>Mix well and incubate in ice water bath for 15 min. Tap the tube gently every 3 min&#160;to maximize binding of biotin-labeled antibodies to their target cells.</li>
		<li>Add 12 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Aspirate supernatant to remove unbound biotinylated antibodies.</li>
		<li>Add 150 &#181;l&#160;of cell sorting buffer and 100 &#181;l&#160;of anti-biotin beads provided in the CD8&#945;Pos DC isolation kit. Mix well and incubate in ice water bath for 15 min.</li>
		<li>Add 10 ml&#160;cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend cells in 1 ml&#160;cell sorting buffer and pass over a fresh LS column according to procedure in steps 3.1.6 through 3.1.8.</li>
		<li>Collect the unlabeled flow through 2 (FT2) and discard the column retentate. This unlabeled fraction is enriched for CD8&#945;Pos and CD8&#945;Neg DCs.</li>
		<li>Pellet the cells in FT2 by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the cells in 1 ml&#160;cell sorting buffer and add 200 &#181;l&#160;of anti-CD8&#945; conjugated magnetic beads provided in the CD8&#945;Pos DC isolation kit.</li>
		<li>Repeat steps 3.2.2 through 3.2.6. The flow through of the column is enriched for CD8&#945;Neg DCs and depleted for CD8&#945;Pos DCs. This is labeled flow through 3 (FT3).</li>
		<li>To elute the CD8&#945;Pos DCs retained in the column, remove the column from the magnet, add 5 ml&#160;of cell sorting buffer and purge the column matrix using the plunger provided with the LS column.</li>
		<li>Pellet the cells by centrifugation at 300 x g for 5 min at 4 &#176;C. Aspirate supernatant and resuspend in 1 ml&#160;cell sorting buffer. To increase the purity, run the eluted cells again through a fresh LS column by repeating steps 3.1.6 to 3.1.8, except discard the flow through and collect retained cells as in 3.2.11.</li>
		<li>To purify the CD8&#945;Neg DCs from the FT3 suspension, pellet the FT3 by centrifugation at 300 x g for 5 min at 4 &#176;C and resuspend in 300 &#181;l&#160;of cell sorting buffer.</li>
		<li>Add 100 &#181;l&#160;of anti-CD11c magnetic beads. Mix well and incubate in ice water bath for 15 min.</li>
		<li>Add 10 ml&#160;cell sorting buffer and pellet cells by centrifugation at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Repeat steps 3.1.5 through 3.1.8. The CD8&#945;Neg DCs are positively selected with CD11c beads and are bound to the column. The flow through can be discarded.</li>
		<li>Elute the cells retained in the column as described in step 3.2.11 to collect the purified CD8&#945;Neg DCs.</li>
	</ol>
	</li>
</ol>
4. Pulsing APCs with Antigens and Cell Transfer
<ol>
	<li>Count the live cells after purification using trypan blue exclusion 21 to distinguish live and dead cells.</li>
	<li>Incubate the cells with the antigen of choice. Use analogues of glycolipid antigen called alpha-galactosyl&#160;ceramide (&#945;GalCer) at 100 nM concentration13. Typically, plate 106 viable cells/well in complete RPMI medium using ultra-low attachment U-bottom 96 well plates to minimize cell attachment. Incubate the cells in a 5% CO2 atmosphere at 37 &#176;C for 1 - 4 hr.</li>
	<li>Harvest cells by gently pipetting several times. Use wide bore pipette tips to minimize cell damage from shear forces. Wash the wells with PBS and pool these washes to maximize cell harvest.</li>
	<li>Pellet the cells at 300 x g for 5 min at 4 &#176;C and resuspend to desired density in PBS. Typically,&#160;inject 1 million cells in 200 &#181;l&#160;per recipient animal. Keep the cells on ice to maximize viability before administration into host animals.</li>
	<li>Inject cells intravenously into mice either through the lateral tail vain or the retro-orbital venous plexus 19. Use a 27 G needle on a 1 ml&#160;syringe, and inject a maximum volume of 0.1 ml&#160;per mouse.</li>
</ol>

<ol><li>Steinman, R. M., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+novel+cell+type+in+peripheral+lymphoid+organs+of+mice.+I.+Morphology%2C+quantitation%2C+tissue+distribution%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.</a> J Exp Med. 137 (5), 1142-1162 (1973).</li>
<li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cells+and+the+control+of+immunity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Dendritic cells and the control of immunity.</a> Nature. 392 (6673), 245-252 (1998).</li>
<li>Dudziak, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+antigen+processing+by+dendritic+cell+subsets+in+vivo%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Differential antigen processing by dendritic cell subsets in vivo.</a> Science. 315 (5808), 107-111 (2007).</li>
<li>Belz, G. T., Nutt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+programming+of+the+dendritic+cell+network%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Transcriptional programming of the dendritic cell network.</a> Nat Rev Immunol. 12 (2), 101-113 (2012).</li>
<li>den Haan, J. M., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constitutive+versus+activation-dependent+cross-presentation+of+immune+complexes+by+CD8%28%2B%29+and+CD8%28-%29+dendritic+cells+in+vivo%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Constitutive versus activation-dependent cross-presentation of immune complexes by CD8(+) and CD8(-) dendritic cells in vivo.</a> J Exp Med. 196 (6), 817-827 (2002).</li>
<li>Carbone, F. R., Kurts, C., Bennett, S. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-presentation%3A+a+general+mechanism+for+CTL+immunity+and+tolerance%5BTitle%5D)+AND+%22Immunol+Today%22%5BJournal%5D)">Cross-presentation: a general mechanism for CTL immunity and tolerance.</a> Immunol Today. 19 (8), 368-373 (1998).</li>
<li>Yamazaki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD8%2B+CD205%2B+splenic+dendritic+cells+are+specialized+to+induce+Foxp3%2B+regulatory+T+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">CD8+ CD205+ splenic dendritic cells are specialized to induce Foxp3+ regulatory T cells.</a> J Immunol. 181 (10), 6923-6933 (2008).</li>
<li>Mukherjee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DEC-205-mediated+antigen+targeting+to+steady-state+dendritic+cells+induces+deletion+of+diabetogenic+CD8%28%2B%29+T+cells+independently+of+PD-1+and+PD-L1%5BTitle%5D)+AND+%22Int+Immunol%22%5BJournal%5D)">DEC-205-mediated antigen targeting to steady-state dendritic cells induces deletion of diabetogenic CD8(+) T cells independently of PD-1 and PD-L1.</a> Int Immunol. 25 (11), 651-660 (2013).</li>
<li>Melief, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-review%3A+Regulation+of+cytotoxic+T+lymphocyte+responses+by+dendritic+cells%3A+peaceful+coexistence+of+cross-priming+and+direct+priming%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming.</a> Eur J Immunol. 33 (10), 2645-2654 (2003).</li>
<li>Bendelac, A., Savage, P. B., Teyton, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+of+NKT+cells%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">The biology of NKT cells.</a> Annu Rev Immunol. 25, 297-336 (2007).</li>
<li>Benlagha, K., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD1d-restricted+mouse+V+alpha+14+and+human+V+alpha+24+T+cells%3A+lymphocytes+of+innate+immunity%5BTitle%5D)+AND+%22Semin+Immunol%22%5BJournal%5D)">CD1d-restricted mouse V alpha 14 and human V alpha 24 T cells: lymphocytes of innate immunity.</a> Semin Immunol. 12 (6), 537-542 (2000).</li>
<li>Brennan, P. J., Brigl, M., Brenner, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invariant+natural+killer+T+cells%3A+an+innate+activation+scheme+linked+to+diverse+effector+functions%5BTitle%5D)+AND+%22Nature+reviews.+Immunology%22%5BJournal%5D)">Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions.</a> Nature reviews. Immunology. 13 (2), 101-117 (2013).</li>
<li>Arora, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+subset+of+dendritic+cells+controls+the+cytokine+bias+of+natural+killer+T+cell+responses+to+diverse+glycolipid+antigens%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.</a> Immunity. 40 (1), 105-116 (2014).</li>
<li>Semmling, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+cross-priming+through+CCL17-CCR4-mediated+attraction+of+CTLs+toward+NKT+cell-licensed+DCs%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell-licensed DCs.</a> Nat Immunol. 11 (4), 313-320 (2010).</li>
<li>Duriancik, D. M., Hoag, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+identification+and+enumeration+of+dendritic+cell+populations+from+individual+mouse+spleen+and+Peyer%27s+patches+using+flow+cytometric+analysis%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">The identification and enumeration of dendritic cell populations from individual mouse spleen and Peyer's patches using flow cytometric analysis.</a> Cytometry A. 75 (11), 951-959 (2009).</li>
<li>Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flt3+ligand+regulates+dendritic+cell+development+from+Flt3%2B+lymphoid+and+myeloid-committed+progenitors+to+Flt3%2B+dendritic+cells+in+vivo%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo.</a> J Exp Med. 198 (2), 305-313 (2003).</li>
<li>Mach, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+dendritic+cells+stimulated+in+vivo+by+tumors+engineered+to+secrete+granulocyte-macrophage+colony-stimulating+factor+or+Flt3-ligand%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand.</a> Cancer Res. 60 (12), 3239-3246 (2000).</li>
<li>Vremec, D., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+purification+of+large+numbers+of+antigen+presenting+dendritic+cells+from+mouse+spleen%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">The purification of large numbers of antigen presenting dendritic cells from mouse spleen.</a> Methods Mol Biol. 960, 327-350 (2013).</li>
<li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manual+restraint+and+common+compound+administration+routes+in+mice+and+rats%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Manual restraint and common compound administration routes in mice and rats.</a> J Vis Exp. (67), (2012).</li>
<li>Reeves, J. P., Reeves, P. A. Unit 1.9, Removal of lymphoid organs. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aReeves%2C+JP+%22Curr+Protoc+Immunol%22">Curr Protoc Immunol</a>. , (2001).</li>
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</ol>

The authors declare that they have no competing financial interests.

Dendritic cells are accepted to be the major professional antigen presenting cells involved in the priming of T cell responses. Their main function is to survey the tissue microenvironment by taking up and processing antigens for presentation to T cells. In order to study the function of specific DC subsets, these need to be isolated in sufficient numbers using an approach that maintains their normal phenotype and functions. Most protocols rely on the isolation of specific DC subset from na&#239;ve mice. Since these cell...

Dendritic cells (DC) were discovered almost forty years ago as the &#34;large stellate (Greek dendron) cell&#34; found in lymphoid organs1. Many studies have shown that DCs are the only antigen presenting cells that can effectively stimulate na&#239;ve T cells2. A major function of these cells is the uptake and presentation of antigens and their efficient processing and loading of these onto antigen presenting molecules. In mouse spleen, DCs can be separated into plasmacytoid and conventional subsets. The plasmacytoid DCs are characterized by low expression of CD11c and high levels of B220 and Gr-1. They are also positive for the surface marker mPDCA1 and secrete type I interferon in response to toll like receptor 9 (TLR9) ligands. The conventional DCs are high for CD11c and MHC class II expression. They can be split into three distinct subsets based on the surface expression of phenotypic markers such as CD4, CD8&#945;, DEC205, CD11b and dendritic cell inhibitory receptor 2 (DCIR2, recognized by the 33D1 antibody) proteins 3,4. The CD8&#945;Pos DCs are also known as cDC1, are positive for DEC205, but negative for myeloid markers such as CD11b and 33D1. The CD8&#945;Neg DCs, also called cDC2, are positive for 33D1, CD11b and CD4 but lack DEC205. The double negative subset (i.e., negative for both CD4 and CD8&#945;) is relatively rare, and is negative for DEC205 and 33D1. It is the least characterized subset and may be a less differentiated form of CD8&#945;Neg DC.
Phenotypic differences in the various DC subsets also extend to their in vivo functions. The CD8&#945;Neg DCs are highly phagocytic and are thought to present exogenous antigen mainly&#160;via MHC class II to CD4 T cells 3. In contrast, the CD8&#945;Pos DCs are specialized for presentation of soluble protein antigen on MHC class I in a mechanism called cross-presentation. The outcome of cross-presentation depends on the activation status of these DCs5, and can either lead to expansion of cytotoxic T cells (CTLs) or development of regulatory T cells 62,7. Targeting of antigen to CD8&#945;Pos&#160;DCs&#160;using anti-DEC205-antibody-mediated delivery results largely in the deletion of T cells8, whereas presentation of antigens derived from infected apoptotic cells induces a strong CTL response 9.
In addition to recognition of peptide antigens, the mammalian immune system has evolved to recognize lipid and glycolipid antigens. These antigens are presented by CD1 molecules, which are MHC class I-like cell surface proteins that exist in multiple related forms in various mammals. In mice, a single type of highly conserved CD1 molecule called CD1d is responsible for presentation of glycolipid antigens 10. The major population of T cells that recognize CD1d/glycolipid complexes is called invariant NKT cells (iNKT cells). These cells express a semi-invariant T cell receptor (TCR) composed of an invariant TCR&#945; chain that is paired with TCR&#946; chains that have limited diversity 11. Unlike conventional T cells that need to proliferate and differentiate to become activated effector T cells, iNKT cells exist as an effector population and start responding rapidly after glycolipid administration 12. Identification of physiologically relevant lipid antigen presenting cells is an active area of research, and several distinct cell types such as B cells, macrophages and DCs have been suggested to perform this function. However, it was demonstrated that the CD8&#945;Pos subset of DCs is the primary cell mediating uptake and presentation of lipid antigens to mouse iNKT cells 13 and glycolipid mediated cross-priming of CD8 T cells 14.
To compare the efficiency of antigen presentation by different antigen presenting cells, a straightforward approach is to transfer different types of purified APCs pulsed with equivalent amounts of antigen into na&#239;ve hosts. Cell transfer experiments of this type are often performed for immunological studies. However, performing transfer studies with ex vivo antigen treated DCs is challenging, since these cells exist as rare populations in lymphoid organs where they constitute less than 2% of total cells15. It is therefore necessary to enhance the development of these cells in donor animals to increase the efficiency of isolation protocols.
It is known that the common lymphoid and common myeloid progenitors, which are required for generation of pDC, CD8Pos and CD8Neg DC subsets, express fms-related receptor tyrosine kinase 3 (Flt-3). Upon in vivo Flt-3 ligand (Flt-3L) administration, emigration of Flt-3 expressing progenitor cells from bone marrow is increased, resulting in the increased seeding of peripheral lymphoid organs and the expansion of their mature DC progeny16. Expression of Flt-3 is lost during commitment to the B, T or NK cell differentiation pathways. Therefore, only minimal alterations are observed in these cells upon Flt-3L administration. Similar expansion in DC populations is observed in mice bearing tumors generated by implantation of a B16-melanoma cell line secreting murine Flt-3L, which provides a simple and economical method for providing sustained systemic levels of Flt-3L17,18. Using this approach, we have developed a protocol based on the implantation of B16-melanoma cells secreting Flt-3L to stimulate the expansion of all normal DC subsets in mouse spleen, thus greatly increasing the yields of these cells that can be isolated for subsequent experiments. We consistently find that within 10 - 14 days following subcutaneous implantation of the Flt-3 secreting tumor, mice develop splenomegaly with marked enrichment of DCs to constitute 40 - 60% of total spleen cells. From these spleens, different DC subsets can be isolated with high purity using standard commercially available cell purification kits that employ subset-specific phenotypic markers.

<table><tbody><tr><td>0.05% Trypsin-EDTA&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>25300-054</td><td></td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CDS019936-250MG</td><td></td></tr><tr><td>Collagenase D&amp;nbsp;</td><td>Roche Diagnostics</td><td>11088858001</td><td></td></tr><tr><td>DNase I (dry powder)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>200 proof ethanol&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>9-6705-004-220</td><td>Used to prepare 70% ethanol</td></tr><tr><td>RBC lysis buffer</td><td>Sigma-Aldrich</td><td>R7757</td><td></td></tr><tr><td>RPMI-1640 medium with L-glutamine&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11875-119</td><td></td></tr><tr><td>DMEM medium with L-glutamine&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11995-073</td><td></td></tr><tr><td>200 mM L-glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>MEM non-essential amino acids&amp;nbsp;</td><td>Life Technologies, Gibco</td><td>11140-050</td><td></td></tr><tr><td>MEM essential amino acids&amp;nbsp;</td><td>Life Technologies</td><td>11130-051&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Life Technologies, Invitrogen</td><td>21985-023</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies, Invitrogen</td><td>15630</td><td></td></tr><tr><td>Phosphate buffered saline (PBS Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; free pH 7.2)</td><td>Life Technologies, Invitrogen</td><td>20012-050</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Life Technologies, Gibco</td><td>14040-182</td><td></td></tr><tr><td>0.5 M Ethylenediaminetetraacetate (EDTA) solution&amp;nbsp;</td><td>Life Technologies</td><td>15575-020</td><td></td></tr><tr><td>Bovine serum albumin (BSA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Fetal calf serum</td><td>Atlanta Biologicals</td><td>S11050&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technologies, Invitrogen</td><td>15140-163</td><td></td></tr><tr><td>Trypan blue (dry powder)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6146-5G&amp;nbsp;</td><td>Prepare 0.08% with PBS</td></tr><tr><td>Plasmacytoid Dendritic Cell Isolation Kit II, mouse</td><td>Miltenyi Biotech</td><td>130-092-786</td><td></td></tr><tr><td>Magnetic beads conjugated with anti-mouse CD11c&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-152-001</td><td></td></tr><tr><td>CD8&amp;alpha;&lt;sup&gt;Pos&lt;/sup&gt; mouse DC isolation kit&amp;nbsp;</td><td>Miltenyi Biotech</td><td>130-091-169</td><td></td></tr><tr><td>Fc-gamma receptor blocking antibody (Clone 2.4G2)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Anti-mouse CD11c-FITC&amp;nbsp;</td><td>BD Biosciences</td><td>553801</td><td></td></tr><tr><td>Anti-mouse CD8&amp;alpha;-PerCP&amp;nbsp;</td><td>BD Biosciences</td><td>553036</td><td></td></tr><tr><td>Anti-mouse B220-PE&amp;nbsp;</td><td>BD Biosciences</td><td>553090</td><td></td></tr><tr><td>1 ml syringes&amp;nbsp;</td><td>BD</td><td>26048</td><td></td></tr><tr><td>23 G1 needle&amp;nbsp;</td><td>BD</td><td>305145</td><td></td></tr><tr><td>100 mm Petri dishes&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>875712</td><td></td></tr><tr><td>Surgical instruments&amp;nbsp;</td><td>Kent Scientific</td><td>INSMOUSEKIT</td><td></td></tr><tr><td>Cell strainer (70 &amp;micro;m)</td><td>BD</td><td>352350</td><td></td></tr><tr><td>Large Petri plates&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Vacuum filtration system (500 ml (0.22 &amp;micro;m))</td><td>Corning</td><td>431097</td><td></td></tr><tr><td>LS columns&amp;nbsp;</td><td>Miltenyi</td><td>130-042-401</td><td></td></tr><tr><td>Magnetic stand MACS separator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>Wide-bore 200 &amp;mu;l pipette tips&amp;nbsp;</td><td>PerkinElmer</td><td>111623</td><td></td></tr><tr><td>Corning ultra-low attachment 96-well plates&amp;nbsp;</td><td>Corning</td><td>CLS3474-24EA</td><td></td></tr><tr><td>6-8 week old female C57BL/6 mice&amp;nbsp;</td><td>Jackson Research Laboratories, Jax</td><td></td><td></td></tr><tr><td>Murine B16.Flt3L melanoma cell line</td><td></td><td></td><td>described by Mach &lt;em&gt;et al.&lt;/em&gt;, 2000</td></tr><tr><td>&lt;strong&gt;Serum free DMEM and RPMI media&lt;/strong&gt;&lt;br/&gt;500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine&lt;br/&gt;5 ml MEM nonessential amino acids (100x, 10&amp;nbsp;mM)&lt;br/&gt; 5 ml HEPES Buffer (1 M)&lt;br/&gt;5 ml L-glutamine (200&amp;nbsp;mM)&lt;br/&gt; 0.5 ml 2-mercaptoethanol (5.5 x 10&lt;sup&gt;-2&lt;/sup&gt; M)</td><td></td><td></td><td>Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need. Filter sterilize the media by passing through a 0.22 &amp;micro;m vacuum filtration system.</td></tr><tr><td>&lt;strong&gt;Complete RPMI and DMEM media&lt;/strong&gt;</td><td></td><td></td><td>Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.</td></tr><tr><td>&lt;strong&gt;MACS buffer&lt;/strong&gt;</td><td></td><td></td><td>Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; Free, pH 7.2) and 2&amp;nbsp;&amp;micro;g/ml 2.4G2 (Fc-Block antibody).</td></tr><tr><td>&lt;strong&gt;FACS staining buffer&lt;/strong&gt;</td><td></td><td></td><td>Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer</td></tr><tr><td>&lt;strong&gt;10x Collagenase D and DNase 1 stock solution&lt;/strong&gt;&lt;br/&gt;
Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; to obtain a solution of approximately 1,000-2,000 Units of collagenase activity per ml.</td><td></td><td></td><td>This 10x stock solution can be prepared ahead of time and stored at -20 &amp;deg;C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use.</td></tr></tbody></table>

an efficient and high yield method for isolation of mouse dendritic cell subsets

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.

The outcome of this procedure relies on the expansion of DC subsets by murine Flt-3L expressed by the implanted melanoma cells. The B16.Flt3L tumor was derived from a C57BL/6 mouse, and should be implanted into animals with that strain background in order to avoid failure of the tumor to become established due to rejection. In some cases, it may be desirable to use genetically modified mice to derive DCs with known defects in signaling pathways or receptors of interest. It is important to...

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 ...

The overall goal of this protocol, is to develop of a high efficiency, high yield method for generating dendritic cell or DCs, that fatefully reflect their in-vivo phenotypic and functional divergence. Dendritic cells exist as great populations in lymphoid organs. Therefore, we use filter ligand to increase the frequency of these essential cells in donor tissues to facilitate that isolation.The main advantage of this technique is that it removes generation of all the DC subsets in suitable numbers for analysis using only the commercially available agents. To harvest flt-3 ligand expressing B16 melanoma cells from a 90%confluent culture, first wash the cells with PBS and incubate the culture with 05%trypsin at 37 degrees Celsius. After 5 minutes, quench the protease activity with 20 milliliters of 4 degrees Celsius complete dima medium and detach the adherent cell model layer with light tapping and gentle pipetting.Collect the cells by centrifugation and count them. Then, re-suspend the single cell suspension to a 1 times to the 8 cells per milliliter concentration in PBS for their subcutaneous injection into C57 black 6 recipient mice. When the tumor reaches two to ten milliliters

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Research

JoVE Journal

Immunology and Infection

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

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

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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.

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This video describes a protocol for the generation of dendritic cells--key antigen-presenting cells that activate T cells--from the bone marrow of an adult mouse.

Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

This video demonstrates a procedure for the adoptive transfer of high salt-treated antigen-presenting dendritic cells (DCs) into mice. Murine CD11c+ dendritic cells (DCs) are exposed to a medium containing high amounts of sodium, forming isolevuglandin (IsoLG)-protein adducts in the cells. These adducts serve as neoantigens and are presented on the surface of DCs. The high salt-treated cells are injected into mice through adoptive cell transfer.

A Method for the High Salt Treatment of Dendritic Cells and their Adoptive Transfer into Mice

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.

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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.

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Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into naïve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

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Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

Learn a high-yield method for isolating mouse dendritic cell subsets using Flt-3 ligand expansion and magnetic bead separation. This protocol achieves >94% purity for functional immunology studies.

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

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